tissuetraining.ppt

1. Tissue Submission
All tissue submitted to the core should
be accurately described on both form and
vial. We can process, embedding and
sectioning without asking questions.

2. Fixation
• When the tissue is separated from blood
supply, it will undergo autolysis. Tissue must
be fixed to preserve morphology, keep the
antigenicity or RNA structure.
• Poor fixation can cause bad morphology,
reduce specificity of IHC or Insitu.
• Over fixation may result in masking of the
antigen or strong non-specific background
staining.

3. • 2% paraformaldehyde (bgal, GFP, RFP),
• 4% paraformaldehyde
• 10% buffered formalin
Fixative

4. Four Factors Influencing Fixation
1. Temperature : 4oC.
2. Size: Idea size: 5X5X3mm
Maximal size: 10X20X3mm.
3. Volume ratio:
Tissue volume : Fixative volume at least 1:20.
Processed in 20 ml glass scintillation vial
4. Time: 1. Remove tissue from body to place the
tissue to fixative. EM 10 min LM 1 hour
2. Duration
Overnight(O/N) or 2 days

5. Fixation for Embryos
• Mouse embryos:
• Up to E14.5 fix whole embryos.
• E15.5 – E16.5 : Peel off skin (if skin is formed)
and cut off head and limbs. Make a open area
in chest and abdominal cavity.
• E17.5- P0: Remove skin and head. Opened
chest and remove rib. Open abdominal part.
After fixation remove limbs and abdominal
part(Detail protocol on our core Web).

6. Fixation for adult mouse
Adult mouse: Hair must be removed. Trim tissue to proper size.
Fat need to be removed.
Heart: Dissect away surrounding fat/connective tissues as much
as possible, then let heart immersed in PBS (RT) and let it beat
for 3-4 minutes in order to pump out residual blood in the
chambers. After that, use syringe to inject some fixative (3-
6ml) into left ventricle, then you can see the fluid come out of
arotic root and vena cava. Leave the heart into fixative.
Lung: Instill the PFA in the tracheal to insufflate the lung and
then incubate the lung in the PFA overnight.

7. Fixation for Adult mouse
Liver: Cut to proper size.
Spleen: Cut to 2-3 pieces.
Kidney: Cut 1/3 off for sagittal section. Cut into 2-3 pieces for cross
section.
Stomach: Wash off the content.
Intestine: Wash off the content. Cut into several pieces for cross
section.
Artery: Remove all fat. Fix it in piece of nitrocellulose paper to make
the vessel straight.
Brain: Fix whole brain. Trim to prober size after fixed and washed.
Skin: Remove hair, Cut longitudinal from back of the mouse.
skin for seeing the hair follicle. Fix it in piece of nitrocellulose paper .

8. For best morphology and staining
results take care to:
• Use high-quality fixative and sufficient amounts of fixative.
• Use sharp razor blade and fine instruments such as scissors
and forceps to handle all specimens.
• Keep embryos in cold PBS when dissecting the embryos.
Put dissected embryos into fixative as soon as possible (no
more than one hour).
• Don’t let samples dry during preparation. Samples should
always be covered by solutions.

9. Tissue Processing
• 1. Wash with PBS 1 min.
• 2. Wash with PBS 30 min X4.
• 3. Dehydrate:
• 30% ethanol in ddH2O for 2 hours.
• 50% ethanol in ddH2O for 2 hours.
• 70% ethanol in ddH2O for 4 hours or O/N.
• 95% ethanol for 3 hour X 2 or O/N.
• 100% ethanol for 1 hour X2.
• Store at -20 oC. ( Samples are ready to submit)
( Adult tissue can be dehydrated into 70% ethanol after
PBS wash.)

10. Group your samples
1.Group samples in same container:
Same age and same genotype samples in same
container
2. Group same tissue on same block
3. Maximize your samples in same block or
same slide
4. Leave a blank row to separate each group

11. Group samples in same block

12. Group samples in same block
Adult organs
Whole Heart Frontal 2-3 same block
Cross 3-4 same block
Half Lung 2-3 same block
Liver
Spleen
Kidney
Intestine
Artery 4-6 same block
Brain
Skin: 4 same block

13. Label your sample
• Use printed label to label vial. Put label on up
part of vial.
• Use color sticker for different Lab. Label vial
with name initials and vial #.
• Use pencil or histology grade permanent
marker to label your samples.
•

14. Submit your sample
Put your samples in a container labeled Samples
For Processing on 4th shelf of the Histology -
20oC freezer.

15. Fill out tissue form
• Write the project name on the form.
No longer need project form
• Histology#: Leave it blank.
• Name of Sample: Use the following format and abbreviations for
this column
For wild type mice. Write: CD1, Black 6, etc.
For transgenic mice. Write: 1. Gene name; 2. Tg; 3. Mut or WT;
For knock out mice. Write: 1. Gene name; 2. KO; 3. -/-, +/- or +/+.
For multiple genotypes. Write: 1. Gene name / gene name; 2. -/-,
+/- or +/+.

16. Fill out tissue form
• Age: For embryo, Write E with the Age. (E12.5)
For adult, Write adult first then age ( Adult 2 M)
• Organ & Pieces: Write organ first then the # of pieces
One piece for each organ.
Heart x3
• Section Orientation & Interested Area:
Orientation: Write Cross, Sagittal, Frontal or maximal area only. Do
not use other term.
Interested Area:
You must specify in detail the area that you are interested.

17. Adult Organs
Maximum area for these organs:
Lung
Liver
Pancreas
Lymph node
Fat

18. Adult Organs
Cross or Sagittal for these organs:
Intestine
Stomach
Bladder
Kidney
Spleen

19. Adult Organs
Heart Cross
Frontal
Brain Frontal
Sagittal
Skin Sagittal
Muscle Cross

20. # of sets, # of slides:
• What is 1 Set?
• 1 set is when we put 1 strip of sections on
each slide. Adjacent sections will be on the
same slide.
• What are Sets?
• To do sets we separate every section. Adjacent
sections will be on adjacent slides.

21. # of sets, # of slides:
• What are 1 Set + 2 Sets?
• 1 Set + 2 Sets are when we put the first
strip of sections on the first slide then skip
two sections and the second strip of
sections goes on a second slide. The
sections that are skipped are placed on
separate slides as sets.

22. # of sets, # of slides:
Generally, 8-12 sets will be used for sets.
Tissue or organs will be sectioned 10 sets.
There will be one or two sections on each
slide.
Multiple organs or the same kind of organ
with different genotypes will be on same
slide.

23. # of sets, # of slides:
• 1 set +2sets
• 1 set +2sets (discard 6-10 sections)
• Adult tissue 10 sets
• Embryos E9.5 8-10 setsx1
• Embryos E10.5-12.5 10-12 setsx2
• Embryos E14.5-PO 10-12 setsx3

24. # of sets, # of slides:
• Under # of Sets or # of Slides you should fill in
more detail information on where to start and
where to stop to pick sections on the slides.
You can use the Atlas of Mouse Development
(Kaufman) as a reference and write down both
page and picture numbers for most of mouse
embryos.

25. # of Sets , # of Slides
• Use Core standard slide#
• C001s
• C010c
• C019f

26. # of Sets , # of Slides
Write C# or page# first then # of sets or # of
slides
( C019c, 10sets )
( p228b-234b, 10sets x2)

27. Submit Tissue Form
• Save tissue form end with two initial (PI first
name and your first name)
TissueForm 34es.doc
• Send the Tissue Form through Email as
attachment to: mlu2@mail.med.upenn.edu and
lancheng@mail.med.upenn.edu
• Write “Tissue Form” on the e-mail subject line.

28. Submission deadline
• The weekly sample submission deadline is
Wednesday
• You will get your slides in 1-3 weeks.

29. Histology Submission
1.
1.Send all histology submission to Lan
and MinMin
2. Write correct email subject on your email
( Tissue form, IHC form, InSitu form)
Other questions should be specified
3. Write priority on email subject and copy
email to your PI If your histology request
is urgent