Immunohistochemical analysis of early passage cultures

Preperation of cell blocks for immunohistochemical analysis

1.     Adherent cells were detached using trypsin and washed with PBS.

2.     The cells were fixed in 4% PFA for 15 minutes on ice and washed twice with PBS.

3.     HistoGel ( Cat no: HG-4000-012, Thermofisher Scientific) was placed on heat block at 65C for 15 minutes.

4.     The cell pellet was resuspended in 100µL of HistoGel and a drop of the suspension was places on parafilm. The cell-HisoGel drop was allowed to solidify for 30 minutes at room temperature.

5.     The resulting specimen was placed in processing cassettes, dehydrated using a Leica TP 1050 tissue processor which exposes samples to a serial alcohol gradient.  

6.     The tissue was then embedded in paraffin wax and 3µm sections were cut from the paraffin embedded sections using a microtome.  Sections were mounted on polylysine slides (VWR, Leicestershire, UK) for staining.

7.     Prior to all staining sections were de-waxed in xylene and rehydrated through decreasing concentrations of ethanol through to water before being washed twice in PBS.

8.     Automated staining on a Leica Bond III staining platform was performed. Slides were incubated with BAP1 primary antibody (Santa Cruz Biotechnology Cat# sc-28383, RRID:AB_626723; 1:150) for 15 min at room temperature. Epitope retrieval was completed using HIER using Leica Bond ER2 (high pH) for 30 min and a Leica Bond Polymer Refine with DAB chromogen detections system used.

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