My lab does histology of brain tissue infrequently, but in large groups. Our last batch was approximately six months ago, and we had about 100 subbed slides (chrom alum) left over after the fact.
Rather than toss the slides, I would like to re-sub them for the current round of histology. Our protocol states that we should 're-wash the slides in the acid bath and re-sub', but nobody knows the details of the acid bath. I have also been told that simply adding another layer of subbing solution on top of the existing one can work.
Could someone please share the contents/protocol of the acid bath to scrub the subbing solution off of slides? Additionally, has anyone had any success with adding another layer of subbing solution on top of an old one?
Thank you,
Nathan
Here is a protocol which has an acid/alcohol bath that you might try http://openwetware.org/wiki/Kubke_Lab:Protocols/Gelatin_Subbing#Acid_alcohol_solution
Nathan, it may depend on the final application to your mounted sections (e.g. only Histo-Morphology: histological-histochemical staining reactions, IHC, IF etc.).
I would give it a try for the "old" batch of subbed slides, at least for 1-5 slides carrying "fresh" section mounts to see and judge whether you could use the "old stuff" (provided they have been stored properly: clean= dust-free, dry, in darkness, @RT. ).
Please cf also the results of query / search by Keyword(s) in RG-QUESTIONS(all) by pasting the URL: https://www.researchgate.net/search.Search.html?type=question&query=Chrom+alum into your browser when logged in to RG.
As of my understanding: your saying "our protocol states that we should rewash the slides in the acid bath...." reminds me that slides in former times were cleaned with chromic acid, do you/did you too? Would you mind to share the / YOUR protocol in this thread...since nowadays one perhaps needs not cleaning slides by acid any more.....Important is absolute cleanness (protein- and/or fat-free, free from any contamination, this also means prevention of bacterial contaminants present either prior to subbing or during mixing, handling, applying the subbing gelatinous solution and finally also due to storage). Deborah has pointed to an interesting and copious source of information...but you might find also other web-sources dealing with the subbing of slides in / with Chromalum / Chrome alum (not exhausting results: e.g. cf. also: http://stainsfile.info/StainsFile/prepare/adhesives/chromegelatin.htm, or, http://www.histosearch.com/histonet/Feb99A/chromegelatin.html, respectively). And, naturally, last but not least question (if you don't mind): why you use Chrome alum subbing slides for your (guessing: paraffin-embedded) brain sections?
Regards and best wishes, WM
Apologize for this additional one: see at least
Susan BACHUS at George Mason University, in RG-thread:
https://www.researchgate.net/post/I_have_encounter_a_problem_in_histology_paraffin_tissue_sectioning
Gelatin-Subbed slides [Chromalum]
WEAR GLOVES! (not to protect your hands, but to protect the slides from RNase from your skin)
· Place slides in racks and soak in hot tap water and soap (e.g. dishwashing soap, or Alconox) for at least 1 hr, but not more than a few hours (as soap could leave residue).
· Rinse thoroughly in hot tap water until suds are completely gone.
· Let sit in 80% ethanol for at least one hr. This solution can be re-used until it turns cloudy. Slides can be left overnight in this solution.
· Rinse in a few changes of deionized water.
· Subbing solution: Dissolve 3.0 g of gelatin (300 bloom swine) in 150 ml deionized H2O by stirring while heating to 50 degrees C. Add 450 ml deionized water to cool, and then dissolve 0.3 g CrK(SO4)2*12H2O (“chrom alum”) in the solution.
· Filter this solution through Whatman Type I filter paper to remove any air bubbles. This solution is only good for the few hrs it will take for this batch of slides, as it will quickly solidify.
· Dip the slides into the subbing solution, drain the slides onto a paper towel and allow to air dry for 1 hr.
· Dip the slides into the subbing solution again. Drain and cover loosely with bench paper and allow to dry for a few hours.
· When thoroughly dry, heat in oven at ~ 50 degrees C for a few hours, then store the slides in slide boxes in zip-lock bags. As long as they are kept dry, they should be good for years.
Thank you very much for that information, Dr. Muss.
In response to your questions, there is no lab protocol for the acid bath, hence my confusion. From what I can tell, it is a case of insider knowledge, that a previous grad student knew the protocol, and thought writing 'acid bath' would be sufficient instruction. As such, I do not know if our slides were cleaned in chromic acid or another substrate.
Our brain sections actually are not paraffin-embedded, but formalin fixed and cut on a freezing stage microtome (at 50 microns). I can say from experience that our method of subbing, at least when the slides are new, are entirely satisfactory when it comes to keeping the tissue adhered.
I will try the additional steps you mentioned. Thank you again for the advice,
Nathan
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