Single Pre-Embedding Immunoperoxidase for Electron Microscopy Sample Clauses

Single Pre-Embedding Immunoperoxidase for Electron Microscopy. After sodium borohydride treatment, selected tissue sections were transferred to a cryoprotectant solution (PB, 0.05M, pH 7.4, containing 25% sucrose and 10% glycerol) for 20 minutes and then frozen in the –80oC freezer for 20 minutes. The tissue was returned to the cryoprotectant solution and then, via a decreasing gradient of cryoprotectant solutions (100%, 70%, 50%, 30%) the tissue was returned to PBS. Brain tissue sections were incubated for 1 hour at RT in PBS containing 10% normal goat serum (NGS), 1% BSA, followed by a 48 hour incubation at 4 degrees with primary antibodies diluted in a solution of normal serum (1%), bovine serum albumin (BSA, 1%), 0.01% Triton X-100 and PBS. The rest of the protocol was the same as for the LM procedure. Following the DAB reaction, the sections were transferred to PB (0.1 M, pH 7.4) for 10 min and exposed to 1% osmium tetroxide diluted in PB for 20 min. They were then rinsed with PB and dehydrated in an increasing gradient of ethanol. Uranyl acetate (1%) was added to the 70% alcohol to increase contrast at the electron microscope. The sections were then treated with propylene oxide before being embedded in epoxy resin (Durcupan, ACM; Fluka, Buchs, Switzerland) for 12 hours, mounted on microscope slides, and placed in a 60°C oven for 48 hours. Blocks of tissue were cut into 60 nm ultrathin sections with an ultramicrotome (Ultracut T2; Leica, Nussloch, Germany) and serially collected on single-slot Pioloform- coated copper grids. The sections were stained with lead citrate for 5 min (Xxxxxxxx, 1963) and examined with a Zeiss EM-10C electron microscope (Thornwood, NY). Fifty micrographs of randomly encountered labeled elements were captured with a GATAN camera at 20,000X from the most superficial sections of blocks of tissue from each of the five animals used in these studies. Labeled and unlabeled neuronal elements were identified and counted.
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