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Author Topic: Problem with keeping tissue on slides.  (Read 2529 times)

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Offline SRajpara

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Problem with keeping tissue on slides.
« on: March 10, 2015, 09:20:35 PM »
Hello all,

I've written in the General Histology section about my problems with H&E, but now it's all cleared up. I'm working on an IHC protocol and I'm experiencing tissue falling off or folding at the primary antibody incubation step. I also kind of noticed the tissue is loose after the antigen retrieval step, so I'm not sure if that's the cause of my problem. I work with super frost plus slides with 8micron thick frozen tissue sections. We store the slides (with sections mounted) at -20C prior to staining. The protocol I use is as follows:

Tissue Adhesion and Antigen Retrieval
1.1.   Bake tissue onto slides by placing selected slides in 55C oven for 1 hour (on aluminum foil)
1.2.   Steam citrate buffer in plastic staining dish for 20 minutes.
1.3.   Add slide(s) to citrate buffer in steamer for antigen retrieval for 20 minutes.
1.4.   Remove staining dish with slide(s) from steamer to cool for 20 minutes.

Blocking
1.5.   Place 2 drops (about 75L) of blocking solution on tissue section(s) on slide(s) and quench for 10 minutes.
1.6.   Wash slide(s) in PBS in plastic staining dish for 5 minutes.
1.7.   Place 2 drops (about 75L) of 2.5% normal horse blocking serum on each tissue section for 20 minutes.

Primary Antibody Staining
1.8.   Incubate tissue sections in primary antibody (1:100 dilution in 1% BSA. Approximately 150uL/tissue section) for 1 hour. 
1.9.   Wash in PBS for 5 minutes.

Secondary (Universal) Antibody Staining
1.10.    Incubate tissue sections in Universal Secondary Antibody for 30 minutes and cover with aluminum foil to shield slide(s) from light.
1.11.    Wash (2x) in PBS buffer for 5 minutes.
1.12.    Incubate in peroxidase substrate solution for 2-5 minutes.
1.13.    Rinse in tap water for 5-10 seconds.

Counterstaining
1.14.    Place 1 drop (about 38L) of Hematoxylin onto each tissue section using a pipette.
1.15.    Immediately rinse in tap water for 5-10 seconds.

Clearing and Coverslipping
1.16.    Incubate slide(s) in Citrisolv for 15 minutes.
1.17.    Mount with ImmunoHistoMount


If anyone has any suggestions, I would really appreciate it. Thank you.

SR

Problem with keeping tissue on slides.
« on: March 10, 2015, 09:20:35 PM »