Question.

What is the maximum thickness that I could use in slide to guarantee the correct penetration of the antibodies and the ABC complex? I usually used 50 um thick sections.

Answer 1.

For sections that thick you would collect them as you always have, stain them as floating sections and then mount onto a glass slide. The routine standard methods for IHC will never penetrate a 50um thick section.

Colleen Forster HT(ASCP)QIHC

Answer 2.

I cannot do my usual free floating staining because this tissue is very brittle. So I was thinking to do the staining in slide at 30 microns?

Alonso

Answer 3.

Even 30um is too thick for the routine IHC on glass slide. The antibody will never penetrate the whole thickness for IHC staining.

Colleen Forster HT(ASCP)QIHC

Answer 4.

Firstly, what antibody( ies) are you wanting to use on your Pwax sections? If you are looking just to identify proteins, 8 microns is sufficient. I mount my brain sections ( human, ms, rat, axolotl, worm, fruitfly, pig...etc) then airdry them O/N in a working fume hood. Then, before dewaxing, I put them into a 60C oven for 1hr. Imho, free-floating sections are useful only if you wish to study projections over depth. Use Superfrost Plus (or equivilent) slides.

Carl Hobbs FIBMS

Histology and Imaging Manager

Wolfson CARD, Guys Campus, London Bridge

Kings College London