Hi,
I want to do immunohistochemistry of GFAP in brain tissue (coronal sections). And this the first time I am doing IHC of mouse brain tissue. Normally for histopathology we take 5 micron sections, but in articles that I referred for IHC, the thickness is considered 30 micron. Please guide me through if the section thickness matters for IHC.
Moreover, I am going to use GFAP Rabbit mAB, provide me general guidance for AB dilution strategy (how much amount of AB solution I need for one slide (3 sections).
Good morning,
The thickness of your slices depends on the technic you use for the storage of your brains. If you freeze them (directly or after embedding in tissuteck) I recommand using thicker slices (30-40µm). If you have paraffin-embedded tissues then you can use a microtom and cut 5-13µm slices without the risk of breaking them.
Best regards
Alicia
Hello Mam,
Good morning,
The thickness depends on the technic you use for the storage of your brains. If you freeze them (directly or after embedding in paraffin wax) I recommend using thicker slices (10-15µm). If you have paraffin-embedded tissues then you can use a microtom and cut 4-8µm.
I agree with previous comments. If you use the frozen brains, 30 μm serial coronal sections is recommended. See Mori et al. - Progress in Neuro-Psychopharmacology & Biological Psychiatry 75 (2017) 94–105 (http://dx.doi.org/10.1016/j.pnpbp.2016.11.005).
Hi Amisha, incase of brain tissue, you need to fix the brain using 4%PFA and if you use frozen free floating sections then 30 micron thickness is the best. You can have a look my articles
http://www.sciencedirect.com/science/article/pii/S0306452216305930
http://www.sciencedirect.com/science/article/pii/S0306452212001728
Hello Amisha,
I completely agree with the comments of the previous respondents. Reiterating them all in summary- using FFPE tissues, 4-6 micra would be very ideal, 30-40 micra for frozen tissue sections. This is the usual thickness for all tissue types, not necessarily for neural tissues only.
You can use the same thickness as you used for histopathology. I am using even thinner sections in liver, intestine and placenta. The thinner;the better.
This thread has been helpful. I am working with frozen brain tissue sections. It is really difficult to get perfect slices 5-10 µm from frozen brain tissues. Usually, at this range, either my tissues fall off the slides after IHC or the integrity is not maintained, tissues break. Today, I will try different section (5 µm, 10 µm, 20 µm, 30 µm, and 40 µm) and proceed IHC and DAB staining. Thanks for the suggestion. The paper from Mori et al., 2017 is insightful.
If your tissue is fixed with 4% paraformaldehyde , the best thing is to do 30/40 µm floating sections. and perform IHC in culture wells. After DAB, you spread them on super-frost+ glass slides and allow them to dry for 1 night before dehydratation and coverslipping.
With these thick sections, you see better the arborization of astrocytes.
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