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 : 4 o C. 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 ddH 2 O for 2 hours. 50% ethanol in ddH 2 O for 2 hours. 70% ethanol in ddH 2 O 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 o C. ( 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. 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 - 20 o C 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 BrainFrontal Sagittal SkinSagittal MuscleCross

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.edumlu2@mail.med.upenn.edu 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