Hello everyone,
I am attempting to stain the trabecular bone of proximal tibia and cortical bone of the midshaft tibia. Tissue was fixed in formalin for approx. 4 months followed by decalcification. Paraffin embedded bone sections (thickness: 4-5 um) are placed on positively charged slides.
I have a trouble getting the sections to adhere to the slides for staining. After cooking in Citrate buffer samples start to come off.
Any suggestions?
Thanks,
Amit
After sectioning, placing and removing water on your slides , place dozens of joseph paper on it, with your hand, spread out vigorously the joseph paper, the same way you stick two papers with glue, remove gently the joseph paper.
Let dry at 37°C Overnight. with this method i never have coming off, the other method is to use glycerin albumen instead of water when you place your sections but i'm always afraid of adding background.
Don't overheat your section when you are placing your sections on your slide i recommend to be at 45 to 50°C 2-5min, else you will completely melt the paraffin and your sample could be disperse
Sorry to ask, after calcification did you process the samples in ethanols to dehydrate before embedding in paraffin wax? Otherwise I agree with Raphael above, dry the slides out overnight either at 37 degrees for 12 hrs or room temperature for 12 hours then 60degrees for 1-2 hours. When heating with Sodium citrate be sure not to allow the liquid to boil too vigorously as this can affect sample adhesion.
FYI You should not fix the samples for so long in Formalin they will likely have antigen damage and not stain as well as they should.
Use artificially available adhesive slides like lysine coated. Dont boil citrate buffer at antigen retrieval step, try to perform this step just below the boiling point and after this step dip your slides in chilled PBS buffer.
Hope this will help, Best of Luck !
Hi Michael,
Yes, these samples were processed in 70% ethanol at least for 24 hrs before paraffin embedding.
Even after fixing in Formalin for longer duration staining is okay.
Thanks!
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