I have 30 um thick cryosections of rat tissue (eye ball). Can this much thickness work for IHC, IF and TUNEL assay ? What optimizations should be done to get as acceptable results as we get in 5-7 um thick cryosections ? Apart from trouble-shooting the staining protocol, do I need to shift from fluorescence microscope (for IHC and TUNEL) to confocal microscope for getting reliable results, considering the thickness of cryosections ?
Hello, I have no experience with section thicknesses for IF and TUNEL, only with IHC. Here, we have already worked with cryosections up to 50 µm thick. A 15 µm thick cryosection shrinks to approx. 4 µm during drying. At 30 µm, it would then be approx. 8 µm. However, I can only assume that it should also work for IF and TUNEL.
Hello,
you can do immunohistochmistry as well as immunfluorescence or insitu hybridization. Ajustment of your immunoprotocol: depending of your fixation (4% PFA or formalin) you can use detergents like Triton X100 in a concentration of 0.2 to 0.5% or Tween 20 (non fixed tissue). I recommend to incubate the primary antibody overnight at room temperature. I recommend also to make a small test serie to optimize the antibody concentration and the incubation time. If you are using double or multiplex labeling then a konfocal microscope is highly recommended because you can run a z-stack to find out weather your signal is insite or outsite of the cell. If you have a fluorescence microscope with optical sectioning like a Zeiss Apotome then you ca use this microscope.
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