Introduction, the principle of immunofluorescence, Technique, Fluorescent microscope and its components, Application and types of immunofluorescence, Direct and indirect immunofluorescence, FACS (Fluorescence-activated cell sorting), Uses and limitations of Immunofluorescence
Immunofluorescence (IF) or cell imaging techniques rely on the use of antibodies to label a specific target antigen with a fluorescent dye (also called fluorophores or fluorochromes) such as fluorescein isothiocyanate (FITC).
Immunofluorescence : Immunofluorescence is a powerful technique that utilizes fluorescent-labeled antibodies to detect specific target antigens..
Fluorescein is a dye which emits greenish fluorescence under UV light. It can be tagged to immunoglobulin molecules.
This technique is sometimes used to make viral plaques more readily visible to the human eye.
Immunofluorescent labeled tissue sections are studied using a fluorescence microscope.
Immunofluorescence (IF) or cell imaging techniques rely on the use of antibodies to label a specific target antigen with a fluorescent dye (also called fluorophores or fluorochromes) such as fluorescein isothiocyanate (FITC).
Immunofluorescence : Immunofluorescence is a powerful technique that utilizes fluorescent-labeled antibodies to detect specific target antigens..
Fluorescein is a dye which emits greenish fluorescence under UV light. It can be tagged to immunoglobulin molecules.
This technique is sometimes used to make viral plaques more readily visible to the human eye.
Immunofluorescent labeled tissue sections are studied using a fluorescence microscope.
Immunodiffusion -Different Types,Principle,procedureand application. it is a diagnostic technique for the detection or measurements of antibodies and antigens by their precipitation which involves diffusion through a substances such as agar or gel agarose .common types -oudin procedure,oakley fulthorpe procedure ,mancini technique ,ouchterlony double immuno diffusion
Enzyme linked immunosorbent assay (elisa) and its clinical significancerohini sane
A comprehensive presentation on Enzyme Linked Immunosorbent Assay (ELISA) and its clinical significance for MBBS, BDS, B Pharm & Biotechnology students to facilitate self- study.
what is sandwich elisa, introduction to elisa, its type and main focus on sandwich elisa, , its process and advantages along with the disadvantages, its applications
Enzyme immunoassays (EIAs), also known as enzyme-linked immunosorbent assays (ELISAs), combine antibody binding with enzymatic detection to quantify molecules of interest.
This method is used to visualise the localisation and quantity of a protein of interest. The target protein is bound to by a specific primary antibody, which in turn is detected by a secondary antibody conjugated to a fluorophore. A fluorescent or confocal microscope is used to visualise the protein.
Immunocytochemistry (ICC) differs from immunohistochemistry (IHC) in that the former is performed on samples of intact cells that have had most, if not all, of their surrounding extracellular matrix removed. In contrast, immunohistochemical samples are sections of biological tissue, where each cell is surrounded by tissue architecture and other cells normally found in the intact tissue. These differences cause the samples to be prepared differently. For ICC, the sample requires permeabilisation so that the antibodies can reach the intracellular targets. Depending on the thickness of the sample, IHC samples do not require this.
Do you have a technical question? Get in touch: info@stjohnslabs.com
Immunodiffusion -Different Types,Principle,procedureand application. it is a diagnostic technique for the detection or measurements of antibodies and antigens by their precipitation which involves diffusion through a substances such as agar or gel agarose .common types -oudin procedure,oakley fulthorpe procedure ,mancini technique ,ouchterlony double immuno diffusion
Enzyme linked immunosorbent assay (elisa) and its clinical significancerohini sane
A comprehensive presentation on Enzyme Linked Immunosorbent Assay (ELISA) and its clinical significance for MBBS, BDS, B Pharm & Biotechnology students to facilitate self- study.
what is sandwich elisa, introduction to elisa, its type and main focus on sandwich elisa, , its process and advantages along with the disadvantages, its applications
Enzyme immunoassays (EIAs), also known as enzyme-linked immunosorbent assays (ELISAs), combine antibody binding with enzymatic detection to quantify molecules of interest.
This method is used to visualise the localisation and quantity of a protein of interest. The target protein is bound to by a specific primary antibody, which in turn is detected by a secondary antibody conjugated to a fluorophore. A fluorescent or confocal microscope is used to visualise the protein.
Immunocytochemistry (ICC) differs from immunohistochemistry (IHC) in that the former is performed on samples of intact cells that have had most, if not all, of their surrounding extracellular matrix removed. In contrast, immunohistochemical samples are sections of biological tissue, where each cell is surrounded by tissue architecture and other cells normally found in the intact tissue. These differences cause the samples to be prepared differently. For ICC, the sample requires permeabilisation so that the antibodies can reach the intracellular targets. Depending on the thickness of the sample, IHC samples do not require this.
Do you have a technical question? Get in touch: info@stjohnslabs.com
Flow cytometry (FCM) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles. In this process, a sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument.
Want to learn about immunofluoresence? This presentation will go over some basic and popular immunofluoresence concepts in a concise fashion. Featuring:
Introduction
History
Similarities & Difference between IF and IHC
Types of Immunofluorescence
Popular Terms
Commonly used Fluorophores
Disease Diagnosed by Immunofluorescence
Antibodies, Proteins and Genes associated with Immunofluorescence
Want to learn about immunofluorescence? This presentation will go over some basic and popular immunofluorescence concepts in a concise fashion.
- Introduction
- History
- Similarities & Differences between IF and IHC
- Types of Immunofluorescence
- Popular Terms
- Commonly used Fluorophores
- Diseases Diagnosed by Immunofluorescence
- Antibodies, Proteins and Genes associated with Immunofluorescence
Immunofluorescence is a technique allowing
the visualization of a specific antigen by
bindIng a specific antibody chemically
conjugated with a fluorescent dye.
Here is some information about 5 important immunological techniques including Flowcytometry and RIA
I hope it helps and please comment if u come across any mistakes or scope for improvement, it'll really be appreciated.
For many years scientists yearned for the possibility of performing flow cytometry to analyse small bio-nanoparticles that are too small to be measured by conventional and high sensitivity instruments. These entities, extracellular vesicles, gene therapy vectors, viruses and drug delivery particles, are promised to become the next generation of therapeutics, but they have been hard to handle and characterise due to their small size and biological or chemical heterogeneity. There is therefore a strong case for bringing flow cytometry capability to the sub-200nm scale.
NanoFCM has developed the NanoAnalyzer platform that now enables true flow-cytometry measurement at the sub-micron scale, and down to particle sizes unreachable by any other flow cytometers (10-40nm depending on the nature of the substrate). Nano-flow cytometry, the technology that underpins the NanoAnalyzer, removes bias and uncertainty stemming from the use of fluorescence signal triggering by using its highly sensitive side-scatter channel to trigger particle events. The single-particle nature of the measurement prevents uncontrolled swarming events, reinforcing data integrity. High resolution of both scatter and fluorescence channels allows the assessment of subpopulations, based on size or on bio-chemical properties.
Nano-flow cytometry’s ability to measure simultaneously a (bio)-nanoparticle population for size, size distribution and bio-chemical properties on a single instrument dramatically improves data quality and throughput compared to the traditional, multiple-techniques approach involving particle characterisation and counting (DLS, NTA, RPS), combined with chemical and biological function assessment (ELISA, Western Blot, Flow Cytometry, PCR). Quantitative measurement of the active and contaminant particles in a single preparation opens up the possibility of characterisation-based nanomedicine regulatory approval, and allows the conduct of large-scale clinical studies. From the research laboratory to the quality control department, NanoFCM delivers comprehensive bio-nano analysis.
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
2. Introduction:
In 1944, Albert Coons showed that antibodies could be labelled with
molecules that have a property of fluorescence.
Fluorescent molecules absorb light of one wavelength and emit light of
another wavelength.
Immunofluorescence is the labeling of antibodies or antigens with
fluorescent dyes, or fluorochrome.
This technique is sometimes used to make viral plaques more readily visible
to the human eye.
Immunofluorescent labeled tissue sections are studied using a fluorescence
microscope i.e the epifluorescence microscope, and the confocal
microscope
Fluorescein is a dye which emits greenish fluorescence under UV light. It
can be tagged to immunoglobulin molecules.
3. The basic principle of
immunofluorescence
To use a fluorescent compound (usually
fluorescein and rhodamine) to detect the binding
of antigen and antibody
The Ab is labelled with the fluorescent
compound
Under a fluorescence microscope, fluorescein
appears bright green and rhodamine appears
orange/red wherever the binding occurs
4. Technique:
Common dyes: fluorescein isothiocyanate (FITC) or tetramethyl
rhodamine isothiocyanate (TRITC)
Dyes chosen are excited by a certain light wavelength, usually blue
or green, and emit light of a different wavelength in the visible
spectrum
Eg. Fluorescein emits green light
Eg. Rhodamine emits orange/red light
Highly fluorescent substances such as phycoerythrin and
phycobiliprotein have also been used.
By using selective filters in a fluorescence microscope only the light
from the dye is detected
Available fluorescent labels now include red, blue, cyan or yellow
fluorescent proteins
5. STOKES FLUORESCENCE:
The phenomenon of fluorescence was first
explained by a British scientist, Sir George
Stokes, in 1852, the shift in wavelength from
short to long during fluorescence is called
“Stokes shift”
Stokes fluorescence is the re-emission of
longer wavelength photons by a molecule that
has absorbed photons of shorter wavelengths.
Both absorption and emission of energy are
unique characteristics of a particular molecular
structure. If a material has a direct band gap in
the range of visible light, the light shining on it
is absorbed, causing electrons to become
excited to a higher energy state.
The electrons remain in the excited state for
about 10-8 seconds. This number varies over
several orders of magnitude, depending on
the sample and is known as the fluorescence
lifetime of the sample. The electron returns to
the ground state and energy is emitted.
6. FLUORESCENT MICROSCOPE
Several microscope designs can
be used for analysis of
immunofluorescence samples.
The specimen is illuminated with light
of a specific wavelength which is
absorbed by the fluorophores,
causing them to emit light of longer
wavelengths
The illumination light is separated
from the much weaker emitted
fluorescence through the use of a
spectral emission filter.
7. Components of a fluorescence
microscope
Light source (xenon
arc lamp or mercury-
vapor lamp)
Excitation filter
Dichroic mirror (or
dichromatic
beamsplitter), and
Emission filter
9. Direct immunofluorescence
Uses:
Direct detection of Pathogens or
their Ag’s in tissues or in pathological
samples
Also used for localization of IgG in
immune complexes along the dermal-
epidermal junction of skin biopsies
from patients suffering from systemic
lupus erythematosus
The aim is to identify the presence and
location of an antigen by the use of a
fluorescent labeled specific antibody
10. Advantages of direct immunofluorescence:
Shorter sample staining times and simpler dual and triple labeling
procedures.
In cases where one has multiple antibodies raised in the same
species, for example two mouse monoclonal, a direct labeling may
be necessary.
Disadvantages of direct immunofluorescence:
Lower signal, generally higher cost, less flexibility and difficulties
with the labeling procedure when commercially labeled direct
conjugates are unavailable
11. Indirect immunofluorescence
The aim is to identify the presence of
antigen specific antibodies in serum. The
method is also be used to compare
concentration of the antibodies in sera.
Indirect test is a double-layer technique,
uses two antibodies i.e the primary
antibody and secondary antibody, which
carries the fluorochrome
The most widely used method of IF in
pathology.
USES:
For the diagnosis of bacterial, viral and
protozoan diseases including:
Borrelia burgdorferi ,
Rickettsia rickettsiae, Rocky Mountain
Spotted Fever,
Bovine immunodeficiency like virus and
Toxoplasma gonadii
12. Advantage over direct IF
The primary antibody does not need to be conjugated
with a fluorochrome because the supply of primary
antibody is often a limiting factor, indirect methods
avoid the loss of antibody that usually occurs during
the conjugation reaction.
Indirect methods increase the sensitivity of staining
because multiple molecules of the fluorescence
reagent bind to each primary antibody molecules,
increasing the amount of light emitted at the location of
each primary antibody molecule.
13. FACS (Fluorescence activated cell sorting)
Fluorescent antibody techniques are extremely valuable qualitative tools,but
do not provide quantitative data, this was remedied by the development of
flow cytometry.
FACS was used to automate the analysis and separation of cells stained
with fluorescent antibody.
The FACS uses a laser beam and light detector to count single intact cells
in suspension.
Cells having a fluorescently tagged antibody bound to their cell surface
antigen are exited by the laser and emit light, an attached computer
generate plots of a no. of cells and their fluorescence intensity.
Use of the instrument to determine which and how many members of cell
population bind fluorescently labeled antibodies called ANALYSIS.
Use of the instrument to place cells having different pattern of reactivity in
different containers is CELL SORTING.
15. FACS now allow the use of multiple fluorescent antibodies.
Highly sophisticated flow cytometers simultaneously analyze cell
populations that have been labeled with two or even three different
fluorescent antibodies.
For e.g if blood sample react with a fluorescein tagged antibody
specific for T-cell, and also with phycoerythrin-tagged antibody
specific for B-cell, the percentages of B and T cell may be determine
simultaneously with a single analysis…
16. Uses of FACS
FACS has multiple uses in clinical and research problems i.e. to
determine the kind and the no. of white blood cells in each
population in patients blood sample, by treating appropriately
processed blood samples with a fluorescently labeled antibody and
performing FACS analysis.
Also used for the detection and classification of leukemia
depends heavily on the cell types involved.
FACS also used for the rapid measurement of T-cell sub-
populations, an important prognostic indicator in AIDS. In this
procedure, labeled monoclonal antibodies against the major T-cell
subtypes bearing the CD4 and CD8 antigens are used to determine
their ratio in patients blood. When the number of CD4 T cells falls
below a certain level, a patient is at high risk of opportunistic
infections.
17. LIMITATIONS OF IMMUNOFLUORESCENCE
PHOTOBLEACHING:
Photochemical destruction of a fluorophores due to the generation of reactive
oxygen species in the specimen as a byproduct of fluorescence excitation.
Can be controlled by
(i) reducing the intensity or time-span of light exposure
(ii) increasing the concentration of fluorophores, or by employing fluorophores
that are less prone to bleaching e.g., Alexa Fluors
AUTOFLUORESCENCE
Only limited to fixed (i.e., dead) cells when structures within the cell are
to be visualized because antibodies cannot cross the cell membrane.
An alternative approach is using recombinant proteins containing
fluorescent protein domains, e.g., green fluorescent protein
(GFP),these proteins allows determination of their localization in live
cells.
18. Epifluorescent imaging of the three
components in a dividing human
cancer cell.
Endothelial cells under the microscope
Yeast cell membrane visualized by
some membrane protein fused with
RFP and GFP fluorescent markers.