Special Histotechniques Lab Manual

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UNIVERSITY INSTITUTE OF APPLIED HEALTH SCIENCES
DEPARTMENT OF MEDICAL LAB TECHNOLOGY
LAB MANUAL
PROGRAM NAME: M.Sc. MLT / Sem-3 / Batch-2021
Subject Name: Special Histological Techniques Including Immunohistochemistry
and Molecular Technology
Subject Code: 21MSH-716
Faculty Name: Attuluri Vamsi Kumar (E13404)
Assistant professor
Department of MLT
Prepared by Verified by Approved by
Attuluri Vamsi Kumar
E13404

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SYLLABUS
LIST OF EXPERIMENTS
Coarse Objectives
This course aims to learn the basic concepts and to get high standard knowledge of histo related to
skin, respiratory, digestive, excretory, and reproductive organ systems
Students will learn about the histo pathologies of various organs of human body
This course aims to learn the basic concepts and to get high standard knowledge of histo related to
skin, respiratory, digestive, excretory, and reproductive organ systems
Subject outcomes
CO1 At the end of the course the candidate should be able to define the structural features of
respiratory, digestive, excretory, and reproductive organ systems.
CO2 To develop an understanding of the anatomical pathologies of various organs of human body
CO3 The candidate should be capable of understanding cytology and histopathology of various
organ systems.Student should be capable of providing optimal pathology services in any
medical set up and develop research aptitude at PG level in the subject.
CO4 At the end of the course the candidate should be able to define the structural features of
respiratory, digestive, excretory, and reproductive organ systems.
Experiment No Experiment Name
1. Demonstration of different fixatives used in Histopathology
2. Demonstration of different Microtome used in Histopathology
3. To demonstrate the activity of enzyme
4. Demonstration of following enzymes activity in a Tissue
5. Demonstration of Laboratory method that uses antibodies
6. Demonstrate the FIC and FITC techniques
7. Demonstration of the technique used to separate DN
8. Demonstration of technique for rapidly producing
9. Demonstrate the Flow cytometer technique

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Course Name/ code Special Histological Techniques / 21MSH-716
Experiment No 1
Title Demonstration of different fixatives used in Histopathology
Master subject coordinator Mr. Attuluri Vamsi Kumar
Department / Institute Department of MLT/ UIAHS
Program M.Sc. MLT
Semester 3
➢ 1. DEMONSTRATION OF DIFFERENT FIXATIVES USED IN HISTOPATHOLOGY
Aim: To demonstrate various fixatives used in histopathology
Materials required:
➢ Reagents (Formalin, Alcohol, buffers, Acetic acid, Ethanol, HgCl, NaCl)
➢ Graded glassware
➢ Weighting machine
➢ Pipettes
➢ Stirrers
➢ PPE
Procedure:
1. Phosphate buffered formalin
Formulation
• 40% formaldehyde: 100 ml
• Distilled water: 900 ml
• Sodium dihydrogen phosphate monohydrate: 4 g
• Disodium hydrogen phosphate anhydrous 6.5 g
• The solution should have a pH of 6.8
• Fixation time: 12 – 24 hours
Recommended Applications
The most widely used formaldehyde-based fixative for routine histopathology. The buffer tends
to prevent the formation of formalin pigment. Many epitopes require antigen retrieval for

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successful IHC following its use. Most pathologists feel comfortable interpreting the
morphology produced with this type of fixative.
2. Formal calcium
Formulation
• Calcium chloride: 10 g
Recommended for the preservation of lipids, especially phospholipids.
3. Formal saline
Formulation
• Sodium chloride: 9 g
This mixture of formaldehyde in isotonic saline was widely used for routine histopathology prior
to the introduction of phosphate-buffered formalin. It often produces formalin pigment.
4. Zinc formalin (unbuffered)
Formulation
• Zinc sulfate: 1 g
• Deionized water: 900 ml
• Stir until dissolved then add –

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Zinc formalin solutions were devised as alternatives to mercuric chloride formulations. They are
said to give improved results with IHC. There are a number of alternative formulas available
some of which contain zinc chloride, which is thought to be slightly more corrosive than zinc
sulfate.
5. Zenker’s fixative
Formulation
• Mercuric chloride: 50 g
• Potassium dichromate: 25 g
• Glacial acetic acid: 50 ml
Gives good nuclear preservation but lyses red blood cells due to the presence of acetic acid. Has
been recommended for congested specimens and gives good results with PTAH and trichrome
staining. Produces mercury pigment which should be removed from sections prior to staining
and can produce chrome pigment if tissue is not washed in water prior to processing. Is an
intolerant agent, so, after water washing, tissue should be stored in 70% ethanol.
6. Helly’s fixative
Formulation
• Potassium dichromate: 25 g
• Sodium sulfate: 10 g
• Immediately before use add –
Considered excellent for bone marrow, extramedullary haematopoiesis, and intercalated discs of
cardiac muscle.

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Produces mercury pigment which should be removed from sections prior to staining and can
produce chrome pigment if tissue is not washed in water prior to processing. Is an intolerant
agent, so, after water washing, tissue should be stored in 70% ethanol. Because of the low pH of
this fixative formalin pigment may also occur.
7. B-5 fixative
Formulation
Stock solution
• Sodium acetate anhydrous: 2.5 g
Working solution, prepare immediately before use
• B-5 stock solution: 20 ml
Despite its mercury content and consequent problems with disposal, this fixative is popular for
the fixation of hematopoietic and lymphoid tissue. It produces excellent nuclear detail, provides
good results with many special stains, and is recommended for IHC. Sections will require the
removal of mercury pigment prior to staining. Tissue should not be stored in this fixative but
placed in 70% ethanol.
8. Bouin’s solution
Formulation
• Picric acid saturated aqueous solution. (2.1%): 750 ml
• Acetic acid glacial: 50 ml
Gives very good results with tissue that is subsequently trichrome stained. Preserves glycogen
well but usually lyses erythrocytes. Sometimes recommended for gastrointestinal tract biopsies,
animal embryos, and endocrine gland tissue. Stains tissue bright yellow due to picric acid.

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Excess picric should be washed from tissues prior to staining with 70% ethanol. Because of its
acidic nature, it will slowly remove small calcium deposits and iron deposits.
9. Hollande’s
Formulation
• Copper acetate: 25 g
• Picric acid: 40 g
• Acetic acid: 15 ml
Dissolve chemicals in distilled water without heat.
Recommended for gastrointestinal tract specimens and fixation of endocrine tissues. Produces
less lysis than Bouin. Has some decalcifying properties.
Fixative must be washed from tissues if they are to be put into phosphate-buffered formalin on
the processing machine because an insoluble phosphate precipitate will form.
10. Gendre’s solution
Formulation
• 95% Ethanol saturated with picric acid: 800 ml
• Fixation time: 4 - 18 hours
This is an alcoholic Bouin solution that appears to improve upon aging. It is highly
recommended for the preservation of glycogen and other carbohydrates. After fixation, the tissue
is placed into 70% ethanol. Residual yellow color should be washed out before staining.
11. Clarke’s solution
Formulation

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• Ethanol (absolute): 75 ml
Has been used on frozen sections and smears. Can produce fair results after conventional
processing providing fixation time is kept very short. Preserves nucleic acids, but lipids are
extracted. Tissues can be transferred directly into 95% ethanol.
12. Carnoy’s solution
Formulation
• Ethanol absolute: 60 ml
• Chloroform: 30 ml
Is rapid acting, gives good nuclear preservation, and retains glycogen. It lyses erythrocytes and
dissolves lipids and can produce excessive hardening and shrinkage.
13. Methacarn
Formulation
• Methanol absolute: 60 ml
• Chloroform: 30 ml
Similar properties to Carnoy but causes less shrinkage and hardening.
14. Alcoholic formalin
Formulation
• 40% Formaldehyde: 100 ml
• 95% Ethanol: 900 ml

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• 0.5 g calcium acetate can be added to ensure neutrality
• Fixation time: 12 - 24 hours
Combines a denaturing fixative with the additive and cross-linking effects of formalin. Is
sometimes used during processing to complete fixation following incomplete primary formalin
fixation. Can be used for fixation or post-fixation of large fatty specimens (particularly breast)
because it will allow lymph nodes to be more easily detected as it clears and extracts lipids. If
used for primary fixation, specimens can be placed directly into 95% ethanol for processing.
15. Formol acetic alcohol
Formulation
• Ethanol absolute: 85 ml
A faster acting agent than alcoholic formalin due to the presence of acetic acid that can also
produce formalin pigment. Sometimes used to fix diagnostic cryostat sections. If used for
primary fixation, specimens can be placed directly into 95% ethanol for processing.
Reference:
https://www.leicabiosystems.com/en-in/knowledge-pathway/fixation-and-fixatives-4-popular-
fixative-solutions/

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Experiment No 2
Title Demonstration of different Microtome used in Histopathology
Program M.Sc. MLT
Semester 3
2. DEMONSTRATION OF DIFFERENT MICROTOMES USED IN HISTOPATHOLOGY
Aim: To demonstrate the working mechanism of microtome
Materials required:
➢ Microtome
➢ Wedge knives
➢ Brushes
➢ Paraffin-embedded blocks
➢ Forceps
➢ Personal protective equipment
o Lab coat or gown
o Fully closed shoes
o Gloves
o Safety glasses as required
Procedure: (Rotatory microtome)
• Validate that NO knife is present in the mount
• Retract knife mount to a safe distance from the clamp and lock
• Place sample into clamp and validate its locking
• Unlock fine feed wheel and lower clamp to be in-line with knife mount
• Unlock knife mount and bring towards the specimen in the clamp
• Unlock sample clamp and adjust angle of surface to be even with knife mount and
relock
• Unlock knife clamp and insert knife from exposed side (no controls or levers)
• Bring knife to validate mounting and lock in place
• Adjust knife to be cutting on one side
• Adjust angle of cutting via the knife mount (~6 °)
• Set thickness of cutting
• Bring sample to be adjacent to knife

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• Expose section waste tray, if present
• Begin sampling of tissue
Rotatory Microtome
Source: http://ilovepathology.com/microtome/
Other Microtomes & their uses:
Base sledge microtome
During sectioning, the specimen is held immobile and the knife glides over the top of it.
Applications : large blocks, hard tissues or whole mounts sections.
Rotary rocking microtome:
The retracting motion is utilized in cryostats to move the tissue block away from the knife on the
upstroke, resulting in a flat face to the tissue block.
Sliding microtome:
In ths type of microtome, during sectioning, the knife or blade remains stationary as the
specimen moves beneath it.
Freezing microtome:
This is equipped with a stage upon which tissue can be quickly frozen using either liquid carbon
dioxide, from a cylinder, or a low temperature recirculating coolant.
The knife is moved whilst the tissue block remains static same as sliding microtome.
Used for cutting thin to semi-thin sections of fresh, frozen tissue
Ultra-microtome:
Used to take Ultra-thin sections of specimens. Specially used in electrom microscopy.
The ultramicrotome comes with either a diamond knife, which is used for most biological ultra-
thin sectioning, or a glass knife, which is commonly used for first cutting.
Cryostat microtome [Cryotome]

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A cryostat is a refrigerated cabinet that houses a specialised microtome and has all of the
cabinet’s controls located outside of the cabinet. Primarily used for cutting sections of frozen
tissue
Principle: The interstitial water in the tissue freezes into ice, making the tissue hard and acting
as an embedding medium.
The freezing agents used include liquefied nitrogen (−190°C) isopentane (2-methylbutane)
cooled by liquid nitrogen (−150°C) , dry ice (−70°C), carbon dioxide gas (−70°C)
Uses :
Rapid production of sections for intra-operative diagnosis
Enzyme histochemistry for labile enzymes
Immunofluorescent methodology
Laser microtome:
The instrument used for non-contact sectioning of biological tissues or materials.
A femtosecond laser, which emits light in the near-infrared spectrum, is used to cut the material.
It is possible to make slices with a thickness of 10 to 100 micrometres.
Can be used for very hard materials, such as bones or teeth
Reference:
1. http://surf.ed.ac.uk/wp-content/uploads/2014/02/SuRF-HIS-005.02COPY.pdf
2. https://www.deakin.edu.au/__data/assets/pdf_file/0003/1037460/Microtome-Basic-
Operation-and-Safety-Procedures.pdf
3. http://ilovepathology.com/microtome/

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Experiment No 3 & 4
Title To demonstrate the activity of enzyme
Program M.Sc. MLT
Semester 3
3 & 4 TO DEMONSTRATE THE ACTIVITY OF ENZYME
Aim: To demonstrate the activity of acid phosphatase in the tissue section.
Principle: ACP belongs to the hydrolytic enzyme, which widely exists in body tissue.
The optimum pH level of its hydrolysis reactions ranges from 5.4 to 6.0. Inhibitors of
enzymes show organs and species differences. The prostate ACP is inhibited by tartaric acid
and fluorides, but not by 0.5% formalin; the liver ACP can be inhibited by the three
inhibitors previously mentioned; and the red blood cell ACP can be inhibited byformalin
and fluorides, but not by tartaric acid. However, in most of the tissues, ACPs can be
selectively inhibited by NaF.
Method: Metal (Pb) Precipitation method.
Preparation of solutions
a. Acetate barbiturate buffer Sodium acetate 9.714 g
Sodium barbital 14.714 g
Distilled water 500 mL
b. HCl (1 mol/L)
c. Incubation buffer
Acetic acid barbiturate buffer 1.7 mL
8.5% NaCl 0.7 mL
0.1 mol/L HCl 3.6 mL
Distilled water 24 mL
3.3% Lead nitrate 0.25 mL
3.2% Sodium β-glycerophosphate 2.6 mL (drops while shaking)
0.6 mol/L MgSO4 (activator) 0.7 mLAdjust pH

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level (0.1 mol/L HCl) to 4.7–4.8.
Procedure:
a. Fresh tissue is fixed with 10% neutral formaldehyde for 12–24 hours or with 95%acetone-
liquid alcohol for 4–12 hours (change the fixative for two times, room temperature)
b. Dehydrate the sections with pure alcohol, clear with xylene, embed with paraffinand then
section
c. The sections are dewaxed and immersed in water.
d. Incubate the sections at 37°C for 2–6 hours. Rinse the sections with distilled water
three times, 2 minutes each.
e. Immerse the sections into 1%–2% ammonium sulfide for 1–2 minutes. Rinse the
sections with distilled water three times, 2 minutes each.
f. Routine dehydration, clearance and sealing.
Observation:
……………………………………………………………………………………………………
……………………………………………………………………………………………………
Result:
……………………………………………………………………………………………………
……………………………………………………………………………………………………
Interpretation of results:
ACP-positive reactions are black.
Control group (omit the substrate or add inhibitors 0.01 mol/L NaF) is negative.
Lead phosphatase method shows ACP activities (marked by arrows) in rat renal tubules.
Note:
a. ACP is a kind of soluble enzyme; cold fixation is preferred.
b. Sediments should not present in liquid preparation process.
c. The pH level of incubation buffer must be accurate, no more than 5.2.
d. Lead ions have inhibitory effects to the ACP, and non-enzyme proteins have non-specific
adsorption to the lead ions, so the incubation time should be shortenedas much as possible.
Reference:
1. https://journals.sagepub.com/doi/full/10.1177/002215549704500614
2. http://jamdsr.com/pdf2b/EnzymeHistochemistryAReview.pdf

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Experiment No 5
Title Demonstration of Laboratory method that uses antibodies
Program M.Sc. MLT
Semester 3
5. DEMONSTRATION OF LABORATORY METHOD THAT USES ANTIBODIES
Aim: To demonstrate the production of hybridoma cells for monoclonal antibody synthesis.
Principle: Hybridoma technology involves immunizing a host animal with an antigen of interest
to elicit an immune response. When a sufficient response is achieved as determined by in
vitro testing, the mature B-cells are harvested from the animal spleens. The B-cells are then fused
with myeloma cells using polyethylene glycol (PEG) to generate an immortalized hybridoma cell
line. Through multiple rounds of screening with the help of HAT medium, a single, pure,
hybridoma positively expressing the antibody against the antigen of interest, as determined by
ELISA, is amplified and monoclonal antibodies are purified.
Procedure:
Step I: Immunization of rabbit or rat and extraction of B-lymphocytes
▪ In order to isolate B-lymphocyte producing certain antibodies, rabbit or lab rat is

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immunized through repeated injection of specific antigen (sheep RBCs)
▪ A sample of B-cells is extracted from spleen of rabbit or rat
Step II: fusion of myeloma cell with B-lymphocytes:
▪ The extracted B-lymphocytes is added to a culture of myeloma cell from bone marrow.
▪ The intended result is the formation of hybridoma cells formed by fusion of B-cell and
myeloma cell.
▪ The fusion is done by using Polyethylene glycol (PEG) or by electrophoration or by using
phages.
Step III: selection of hybridoma cell
▪ The next step is selection of hybridoma cells.
▪ The B-lymphocytes contains HPRT1 gene which codes for enzyme Hypoxanthine-guanine
phosphoribosyltransferase (HGPRT). The enzyme HGPRT involved in synthesis of
nucleotides from Hypoxanthine present in culture medium. Therefore B- cells can grow in
medium containing Hypoxanthine amonopterin thymine (HAT media).
▪ But myeloma cell lack HPRT1 gene so, it does not produce HGPTR enzyme and it does
not grow in HAT medium.
▪ The myeloma cell fused with another myeloma cell or those do not fused at all die in HAT
medium since they do not utilize Hypoxanthine.
▪ Similarly, B- cell that fuse with another B- cell or those do not fuse at all die eventually
because they do not have capacity to divide indefinitely,
▪ So, only hybridoma cell ie. fused cell between myeloma and B-cell can survive and divide
in HAT medium.
▪ Screening is done to select hybridoma cells which are the desired cell for monoclonal
antibodies production.
Step IV: culture of Hybridoma cell:
▪ The selected hybridoma cells are cultured in suitable culture medium, often supplemented
with insulin, transferon, ethanol, amine and other additional hormones.
▪ Some commonly used culture media for hybridoma cell for production of monoclonal
antibodies are:
▪ DMEM (Dulbecco’s modified eagle medium)
▪ IMDM (Iscove’s Modified Dulbecco’s Medium)
▪ Ham’s F12
▪ RPMI 1640 medium (Roswell Park Memorial Institute 1640 medium)
Step V: Inoculation of hybridoma cell into suitable host
▪ These hybridoma cells are then injected into lab animal so that they starts to produce
monoclonal antibodies.
▪ These hybridoma cells may be frozen and store for future use.
Step VI: extraction and purification of Monoclonal antibodies:
▪ Monoclonal antibodies from host animal is extracted and purified by one of the following
methods;
▪ Ion exchange chromatography
▪ Antigen affinity chromatography
▪ Radial immunoassay

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▪ Immune precipitation
Application of Monoclonal antibodies:
1. Disease diagnosis:
Once monoclonal antibodies for a given substance have been produced, they can be used
to detect the presence of this substance. Proteins can be detected using the Western blot and
immuno dot blot tests. In immunohistochemistry, monoclonal antibodies can be used to
detect antigens in fixed tissue sections, and similarly, immunofluorescence can be used to
detect a substance in either frozen tissue section or live cells.
▪ ELISA to test HIV, hepatitis, Herpes etc
▪ RIA- to test viral infection
▪ Mabs to Hunam chorionic gonadotropin
Reference:
1. https://www.ibtbioservices.com/assays-and-protein-expression/production-of-
monoclonal-antibodies/
2. https://www.onlinebiologynotes.com/monoclonal-antibodies-mabs-production-by-
hybridoma-technology-application-and-types/
3. https://microbeonline.com/monoclonal-antibodies-types-and-applications/
4. https://en.wikipedia.org/wiki/Monoclonal_antibody

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Experiment No 6
Title Demonstrate the FIC and FITC techniques
Program M.Sc. MLT
Semester 3
6. DEMONSTRATE THE FIC AND FITC TECHNIQUES
Aim: To demonstrate the target antigen in the given tissue section by Fluorescein isothiocyanate
(FITC) method.
Principle: Immunofluorescence utilizes the specificity of antibodies with fluorescent dyes to
recognize their antigen, and therefore allows visualization of the distribution of the target molecule
through fluorescent dyes with a fluorescence microscope. Immunofluorescence is widely used as
an example of immunostaining and is a specific example of immunohistochemistry that makes use
of fluorophores to visualize the location of the special biomolecules. There are two classes of
immunofluorescence techniques including direct and indirect.
Materials
1. 10 Phosphate buffered saline (PBS) (pH 8.0).
2. Formaldehyde: 16%, methanol free, use fresh, store opened vials at 4°C in dark, dilute in
PBS for use.
3. Blocking Buffer: (1% PBS with 5% normal goat serum and 0.3% Triton X-100): add 2.5
ml 10X PBS, 1.25 ml normal serum from the same species as the secondary antibody (e.g.,
normal goat serum, normal donkey serum) and 21.25 ml ddH2O and mix well.
4. Antibody Dilution Buffer
5. Primary antibody and secondary antibody.
6. Prolong Anti-Fade Reagent.
7. Tissue or cell samples
Immunofluorescence protocol for Tissues
For immunofluorescence labelling of organ tissue, a paraformaldehyde fixation and paraffin
embedding protocol is used. The organs are obtains by dissection.
1. The dissected organs are then allowed to fully adhere to the surface of the slides by gently
removing all of the PBS as well as the incubation chamber.

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2. Immediately before the organs are dehydrated, slides are rapidly immersed in cold (-20°C)
acetone for 5 min.
3. PBS containing 1% Triton X-100 is added to the slides, and a PAP pen ring is applied
around the tissue to minimise the volume of reagents.
4. Slides are incubated overnight at 4°C in a humidified chamber made from a Petri dish and
PBS-wet paper.
5. The next morning, slides are washed three times in PBS and used for immunolocalization.
6. Slides are incubated for 30 min in a blocking buffer (PBS, 0.1% Triton X-100, 10% normal
goat serum), then incubated for 2.5 h with primary antibodies which against the target
protein. The primary antibodies are applied to the slides diluted follow the manufacturer’s
recommendation.
7. Slides are then washed three times in PBS and incubated with secondary antibodies.
Depending on the type of primary antibody used, the secondary antibody is labeled with
different fluorescence groups. Secondary antibodies are applied following the
manufacturer’s recommendation, too.
8. Slides are washed three times in PBS, rinsed in water and mounted with ProLong® gold
anti-fade mounting medium containing 4’,6-diamidino-2-phenylindole (DAPI) to
counterstain the cell nuclei.
9. The slides are then visualized under a fluorescence microscope.
Observation
……………………………………………………………………………………………………
……………………………………………………………………………………………………
Result
……………………………………………………………………………………………………
……………………………………………………………………………………………………
Results interpretation
If there is the presence of a specific antigen or antibody of interest they would form an Ag-Ab
complex. So the fluorochrome-conjugated antibody will remain bound in the preparation even
after washing and fluorescence of yellow-green or green or red ( depending on the types of
fluorochromes used ) can be observed while visualizing through a fluorescent microscope. And
the test can be considered positive.
If there is no presence of antigen or antibody of interest then Ag-Ab complex won’t be formed and
all the unbound antibodies would be washed away hence we cannot observe fluorescence if the
test is negative.

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Applications
1. Immunofluorescence can be used on tissues or cell sections to determine presence of
different biological molecules which also includes proteins, carbohydrates, etc.
2. Also used in molecular biology for visualization of cytoskeletons such as intermediate
filaments.
3. It also plays a key role in the detection of autoimmune disorders.
4. It can be used with some non-antibody methods of fluorescent staining, like the use
of DAPI (4′,6-diamidino-2-phenylindole ) to label DNA.
Reference:
1. https://www.creativebiomart.net/resource/principle-protocol-immunofluorescence-
345.htm
2. https://microbenotes.com/immunofluorescence/

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Experiment No 7
Title Demonstration of the technique used to separate DNA
Program M.Sc. MLT
Semester 3
7. DEMONSTRATION OF THE TECHNIQUE USED TO SEPARATE DNA
Aim: To separate DNA form the blood sample by salting out method.
Principle
The salting out procedure is a simple and less expensive method to precipitate the genomic DNA
from blood samples. The isolated DNA will be stored at -80℃ and use for immunogenic studies.
The process of "salting out" is a purification method that relies on the basis of protein
solubility. It relies on the principle that most proteins are less soluble in solutions of high salt
concentrations because the addition of salt ions shield proteins with multi-ion charges. Those
charges help protein molecules interact, aggregate, and precipitate. The exact concentration
resulting in precipitation varies from protein to protein, allowing for the separation of different
proteins (as proteins will precipitate at different points with increases in salt concentration). Salting
out can also concentrate dilute solutions of proteins; once the protein precipitates, the remaining
liquid can be removed. However, the salt can pose a problem to the purity of protein.
Materials required
1) 50ml Falcon tubes
2) 15ml Falcon tubes
3) 1.5ml eppendorf
4) Nitrile Gloves (powder free)
Reagents
1. Red blood cell lysing buffer (RCLB),
2. White blood cell lysing buffer (WCLB),

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3. Proteinase K (20mg/ml) stock,
4. 10% SDS,
5. 3M sodium Acetate PH (5.2),
6. Isopropanol,
7. 80% ice-cold ethanol,
8. Tris EDTA (TE : PH : 8) buffer.
Equipment’s
1. Bench top refrigerator centrifuge (eppendorf),
2. Laminar hood (NUAIRE, biological safety cabinet, Class II),
3. Fridge,
4. Freezer - 30 °C ,
5. Freezer - 80 °C ,
6. Water bath (Thermo scientific),
7. Vortex machine (Genie 2)
Reagent Preparation
Red blood cell lysing buffer (RCLB)
1 liter
1. 10mM TrisHCl = 1.21 gram (PH : 7.6)
2. 10mM NaCl = 0.586g
3. 5mM MgCl = 1.014
The above reagents were dissolved and the pH is adjusted to 7.6 with NaOH then finally makeup
to 1 liter with Distilled water.
White blood cell lysing buffer (WCLB)
1 liter
1. 10mM Tris HCl = 1.21g
2. 10mM EDTA = 3.72g
3. 50mM NaCl = 2.92g
4. SDS agar = 2.0g
These are dissolved and the pH is adjusted to 7.6 with NaOH then finally makeup with 1 liter
distilled water.

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TE Buffer
1 liter
1. 10ml Tris HCl = 1.21g
2. 1mM EDTA = 0.372g
These are dissolved and the pH is adjusted to 8.0 with NaOH then finally makeup with 1 liter
distilled water.
3M Sodium acetate
24.7g sodium acetate was dissolved in 100ml distilled water then pH was adjusted to the pH 5.2
with glacial acetic acid.
80% Ice cold ethanol
80ml of absolute ethanol was mixed with 20ml of nuclease free water and stored at 4℃ condition
10% SDS
10g of SDS dissolved in 100ml of distilled water. Stored at room temperature.
Method
It is a Two day procedure:
DAY-1
A 2-3 ml of blood is suspend in 20ml of RBC cell lysing buffer (RCLB) Then vortex and
incubate at room temperature for 10min. Then centrifuge the tubes at 2000rpm for 15min at
4℃ . Then carefully discard the supernatant without disturbing the cell pellet. Re-suspend with
15ml of RCLB and vortexed and centrifuge at 2000rpm for 15min at 4℃. Then the supernatant
was discarded and 15ml of RCLB were added, vortexed and centrifuged at 2000rpm for 15 at
4℃. Now to the clear cell pellet, add 3ml of white blood cell lysing buffer ( WCLB), 25ᶙl
Protinase K and 50ᶙl of 10% SDS were added. Then the tubes were vortexed for few seconds
and incubated at 56℃ for 1 and half hours in water bath. After incubation the tubes were kept
at room temperature for over night.
Day 2

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Now 4ml of 3M sodium acetate was added to the tubes kept overnight, and Vortexed
moderately for few seconds. Then the tubes were centrifuged at 2000rpm for 15min at 4℃.The
supernatant was transferred to fresh 15ml Falcon tube and equal volume of isoproponal was
added. Then the tubes were gently shaked to precipitate the DNA. The precipitated genomic
DNA was transferred to 1.5ml Eppendrof tube using 1ml pipette. The DNA was pellet down
by centrifugation at 8000rpm for 15min at 4℃. Then the supernatant was carefully discarded.
The pellet is washed with 500ᶙl of 80% ice cold ethonol by centrifuging at 8000rpm for 5min
at 4℃. The supernatant was discarded and again added 500ᶙl of 80% ice cold ethanol was
added and pellet down by centrifuging at 8000rpm for 5min at 4℃. The pellet was allowed to
dry at room temperature ( approximately half an hour by inverting the tubes on clear tissue
paper). Finally add TE buffer based was added based on the pellet size and stored at -80℃ until
use.
Observation
……………………………………………………………………………………………………
……………………………………………………………………………………………………
Result
……………………………………………………………………………………………………
……………………………………………………………………………………………………

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Reference:
https://www.bioted.es/1-1-extraction-and-purification-of-nucleics-acids/

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Experiment No 8
Title Demonstration of technique for rapidly producing DNA
Program M.Sc. MLT
Semester 3
8. DEMONSTRATION OF TECHNIQUE FOR RAPIDLY PRODUCING DNA
Aim: To demonstrate the amplification of DNA by PCR.
Principle
PCR is a method of making multiple copies of a DNA sequence, involving repeated
reactions which uses the enzyme DNA polymerase that directs the synthesis of DNA from
deoxynucleotide substrates on a single-stranded DNA template. DNA polymerase adds
nucleotides to the 3` end of a custom designed oligonucleotide when it is annealed to a longer
template DNA. Thus, if a synthetic oligonucleotide is annealed to a single-stranded template that
contains a region complementary to the oligonucleotide, DNA polymerase can use the
oligonucleotide as a primer and elongate its 3` end to generate an extended region of double
stranded DNA.
Materials
1. Millipore water
2.Taq DNA polymerase Buffer – 10x
• 100mM Tris ammonium per sulphonic acid (TAPS) pH (8.8)
• 500mM potassium chloride
• 15mM Magnesium chloride
• 0.1% gelatine
3. Deoxyribonucleotide tri phosphates (dNTPs) (10mM) (250µmol/1µ of the stock in 1mM Tris
HCl – pH 7.5)

27
• 2.5mM dATP
• 2.5mM dCTP
• 2.5mM dTTP
• 2.5mM dGTP
4. Primers as ( as lyophilized powder )
• final concentration – 10 picomoles
• Forward Primer
• Reverse primer
5. Taq DNA polymerase enzyme
6. Storage and dilution buffer for the enzyme
• 20mM tris (pH 8)
• 100mM potassium chloride
• 0.1mM Ethylene diamine tetra acetic acid
• 0.5 % Tween 20 (v/v)
• 0.5% Igepal
• 50% Glycerol
7. Thermal cycler ( DNA Engine Tetrade, MJ research . Model: PTC 225)
Method
The laminar hood was cleaned with alcohol or with excess of Sodium Hypochlorite
solution. The total volume ( sample + reagents ) per vial used was 25µl. All reagents were
thawed before use but were kept in cold conditions during usage.
The following reagents were added to a sterile eppendorf tube (1ml) in the following order:
• Millipore water - 17.3µl
• 10x buffer - 2.5µl
• Forward primer - 1 pico mole
• Reverse primer - 1 pico mole
• dNTPs - 1mM
• Taq polymerase enzyme - 0.2µl (1 unit )
A master mix was prepared using the following components. 23µl of master mix was then
aliquoted to different eppendroff tubes. 2µl of the sample was added to the reaction mixture in the
vial after spinning it down in a mini centrifuge. Then the vial was placed in the PCR machine. The

28
PCR machine ( Programmable Thermal controller ) was programmed specifically for CYP27B1
gene amplification for the following conditions
• DENATURATION at 95°C for 5 minutes
• ANNELING at 62°C for 45 seconds
• EXTENSION at 72°C for 45 seconds
A final extension at 72°C was provided to ensure complete annealing of DNA. 35 cycles
was performed based on the above conditions. After two and half hours, the vials was taken out
and the product was analysed by 1.5% agarose gel electrophoresis to confirm the amplification of
the CYP27B1 gene.
Plate 2. PCR reagents
A. Milli Q water E. Forward primer
B. dNTP mixture F. Reverse primer
C. 10 x Buffer 1- 8. Alliqoate tubes
D. Taq Polymerase
Plate 3. Themal cycler
DNA Engine Tetrade, MJ
Research . Model: PTC 225
Plate 4. Preparation of PCR master mix

29
Experiment No 9
Title Demonstrate the Flow cytometer technique
Program M.Sc. MLT
Semester 3
9. DEMONSTRATE THE FLOW CYTOMETER TECHNIQUE
Aim: To demonstrate the working mechanism of flow cytometer
Principle: Flow cytometry (FCM) is a technique which enables rapid analysis of statistically
significant number of cells at single cell level. The main principle of this technique is based
on scattering of light and emission of fluorescence which occur when a laser beam hits the cells
moving in a directed fluid stream
Working principle of Flow cytometry (Abbreviations – FL Fluoresce labelled, SSC- Side scattering, FSC –
Forward scattering)
Procedure:
Sample Preparation
• Before running in the flow cytometers, the cells under analysis must be in a single-cell
suspension.
• Clumped cultured cells or cells present in solid organs should first be converted into a

30
single cell suspension before the analysis by using enzymatic digestion or mechanical
dissociation of the tissue, respectively.
• It is then followed by mechanical filtration should to avoid unwanted instrument clogs
and obtain higher quality flow data.
• The resulting cells are then incubated in test tubes or microtiter plates with unlabeled or
fluorescently conjugated antibodies and analyzed through the flow cytometer machine.
Antibody Staining
• Once the sample is prepared, the cells are coated with fluorochrome-conjugated
antibodies specific for the surface markers present on different cells. This can be done
either by direct, indirect, or intracellular staining.
• Indirect staining, cells are incubated with an antibody directly conjugated to a
fluorophore.
• In indirect staining, the fluorophore-conjugated secondary antibody detects the primary
antibody
• The intracellular staining procedure allows direct measurement of antigens presents
inside the cell cytoplasm or nucleus. For this, the cells are first made permeable and then
are stained with antibodies in the permeabilization buffer.
Running Samples
• At first, control samples are run to adjust the voltages in the detectors.
• The flow rates in the cytometer are set and the sample is run.
Clinical significance:
Flow Cytometry is used in several fields including molecular
biology, pathology, immunology, virology, plant biology, and marine biology. Some of the
common application include:
• It is used in clinical labs for the detection of malignancy in bodily fluids like leukemia.
• Cytometers like cell sorters can be used to separate the cells of interest in separate
collection tubes physically.
• It can be used for the detection of the content of DNA by using fluorescent markers.
• Flow cytometers allow the analysis of replication cells by using fluorescent dye for four
different stages of the cell cycle.
• Acoustic flow cytometers are used in the study of multi-drug resistant bacteria in the
blood and other samples.
• The different stages of cell death, apoptosis, and necrosis can be detected by flow

31
cytometers based on the differences in the morphological and biochemical changes.
Reference:
https://microbenotes.com/flow-cytometry/

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