Hello, sorry to hear about your trouble. Once a few years ago, we sliced 7 day old rat brains to 30um. The animals were not perfused fixed but after slicing were put in 4% PFA for about 15 minutes at the start of the staining protocol. 

Please look at our article about rat brain .

You could try using Poly-lysine on the slides to help attach the sections.  Otherwise, there are some free floating section methods.

Dear Sourish Mukhopadhyay,

for now only questions regarding your perfusion fixation (only if you have to perform another " round " (=more puppies) of brain perfusion fix-preps): you wrote:

"...perfused pups with 0.9% saline and then 4% PFA, ...". so for me this is flushing the microvascular system by physiologic saline and guessing, FA was regularly buffered (if yes, which buffer, pH, tonicity?) . It could be that flushing before fixation with 0.9%NaCl-solution (T?) makes some troubles, as well as I think that for puppies' brains 4% FA might be too high percentage. Duration of perfusion with fixative (a well as temperature?) would be also interesting for thinking about. Did you immerse the brains after perfusion into fixative again (t and T?) and did wash then with appropriate buffer solution(s) prior to cryoprotection of brains with 30% sucrose-solution? (You wrote sucrose-PFA, so it really was PFA = paraformaldehyde! with sucrose added? Then frozen and cryosectioned (whole brains)?

Thanks for eventually commenting on these your processing steps.

Deborah - yes, i intend to try the poly-lys coated slides.

Hello Dr. Muss

Thank you for going through my query. I will try to clear/explain my processing steps pointwise.

1. Yes, the pups were flushed with physiological saline, majorly to clear the vascular system of the blood. As is clear, I had performed transcardial perfusion in these animals. The PFA was buffered. It was made in 0.1 M PB solution. 

2. The 4 day old pups weighed approximately 10-13 gms. I used atleast 20 ml of PFA per animal. The bodies had become rigid by the end of this, and when I removed the brains, they had cleared of blood, which means the saline and PFA must have gone through the blood vessels there.

3. I do not think the 4% PFA should be very problematic, going by papers that have described perfusions in animals of the same range. However, i could try to go with a lower percentage and see what happens.

4. Regarding temperature, the perfusions were all performed on ice, with ice cold saline and PFA.

5. The duration of postfixation, from what I understand should affect my immunohistochemistry quality, specifically, it may affect epitope. However, this is a problem with the quality of sections. In any case, initially, I had kept the brains in 4% PFA for postfixation for rather long - around 2-4 weeks, before immersing them in 30%sucrose in 4%PFA, letting the brain 'sink', and subsequently sectioning them.

Let me know if anything strikes you from this information.

Dear Sourish,

thanks for your kind reply. Apologize if my reply might replicate/reiterate your knowledge or is too long for the task here. But as always: I don’t want to „teach“ anybody, least of all in a Forum like RG. As of my understanding (I know this might be “old junk” as my grey cells are…. having done perfusion fixation of rat brains – rats of different ages - also in my student’s years in the 1970ies):

I agree that 4% PFA vs. 2% PFA might not be followed by artificial “morphology”, but since PFA / FA as such in fact is a a higher PFA concentration in puppies’ brain (not as “dense” as old age and containing more water) perhaps could be followed by “much more hardening of tissue” than expected (which means possibly a kind of soft barrier to penetration of [all] solutes during further specimen processing). If I remember correctly I always found microvessels of brain having contracted due to perfusion at 4°C (without maintaining pressure and use of dilatants in the perfusion solution (ok, the flushing with 0.9%NaCl prior to fixation might have washed out most if not all the RBC’s/”blood”, so you “see” the brain as being “cleared from blood”) and therefore “postfixation” by immersing the whole brains in the fixative for some hour might not penetrate and hit ALL regions until the center easily (yes, I agree 2-4 weeks in the fixative definitely will be too long with regard to immuno… but an overnight-immersion at 4°C nevertheless I would try, despite I take your point 5. seriously in any direction you mentioned there).

Also, I thought, there might be some problem with leaving the fixative IN the proper perfused brain and then immersing it into the sucrose solution for cryoprotection. You stated in your request: “…the sections are extremely fragile, and most of them fail to survive the staining…” and I am interpreting this as either poor of brain tissular elements due to kind of “poor” fixation (despite the bodies have been found to “be rigid”, (in literature I have found too: “immersion over night in PFA is fine for mouse brain fixation, especially if the mice were perfused.”) or, thinking in “physical” terms, poor cryopreservation (no full penetration of cryoprotectant) and/or poor (= inadequate) freezing of the whole brain(s) inducing ice crystals within the tissue.

I have seen a lot of recipes, most of them signalize “washing” the specimens after fixation in PBS or an appropriate PO4-buffer

only as one example: cf.

http://medschool.creighton.edu/fileadmin/user/medicine/Departments/Biomedical_Sciences/CUIBIF/tech_gallery_PDFs/Embedding_Protocol.pdf.

Also, it might induce problems to immerse “only” in 30% Sucrose solution (you combine this with additional 4%PFA-solution if I interprete correctly) instead of doing this at least stepwise in increasing concentrations (10,15/20% and 30% Sucrose). Others use 15% Sucrose solution as the highest cryoprotection solution (since 30% may have “gumming up” properties on sectioning). It also has been reported that freezing the brains in the cryostat is not recommended; instead, freezing should be done using precooled isopentane (use dry ice –absolute alcohol bath, wait until temp. reaches 60°-65°C; precooling can be done also in a low temp. refrig. @ -80°C).

Another recipe that has been questioned in an other RG-thread in 2013 (cf. https://www.researchgate.net/post/Cryoprotection_with_sucrose_30_after_neutral-buffered_formalin_NBF_fixation) nearly addresses a similar matter.

Another thread to be recommended would be: https://www.researchgate.net/post/What_is_the_best_fixative_for_cryosectioned_tissues_that_can_maintain_DNA_and_protein_stability

Hopefully I am not wrong with assumption that you “embed” your brains in a medium like OCT prior to sectioning….if you use OCT you should wick/blot the wet sucrose-immersed tissue carefully but thoroughly from any adhering sucrose since otherwise OCT might not bound/adhere to the tissue properly. If not using OCT (i.e. just only a bit “glueing” the object to the plate) , dry edges of the specimen could induce improper sectioning too. Another reported improvement (brain(s)) was

Sourish Mukhopadhyay did you ever find a solution to this? I ask because I plan to perfuse PD4 rats and perform IHC on free-floating brain tissue, but I am currently doing IHC with PD8 tissue and even at that age I am struggling with the tissue's fragility during staining procedures