Hi there! I'm hoping to to look at co-localisation of cytokines and markers of different brain cell types (e.g., neurons, astrocytes, microglia) in juvenile rat brains (postnatal day 28) that have been embedded in paraffin. Ideally, I would like to be able to image co-localisation, and also to analyse the morphology of glial cells that show co-localisation with cytokines.
I was wondering if anyone has any experience with the effects of different tissue slice thicknesses on the effectiveness of immunolabelling and morphological analysis. Most of the studies that do double/triple staining with paraffin-embedded tissue seem to use 3-5 micrometers, but the studies that perform morphometry (and only stain for 1 marker) seem to use thicker slices, e.g., 20-40 micrometers. Are thicker slices of paraffin-embedded tissue harder to stain for multiple markers?
Any advice would be greatly appreciated!
Hello, for immunolabelling, I always use a 5 microns. Increasing the section thickness causes me to use more antibody, which is not ideal. I have read papers about conventional histochemistry techniques that use about thicknesses of 20, 40 and even more, but I do not use and have never tested these thicknesses. I usually 7 microns as limit. Greater thickness can make it difficult for the reagent penetration in tissue, which takes more time for stained, depending on the composition of tissue, overstaining can occur. I perform morphological and morphometric analysis in both techniques without hardness to thicknesses 7 and 5 microns .