Mouse anti-human serum albumin monoclonal antibody and rabbit anti-human PTEN polyclonal antibody were purchased from Santa Cruz Biotech Inc. Human hepatoma PLC/PRF/5 cells were fixed in ice-cold paraformaldehyde solution (4%) followed by soaking in 0.3% Triton-X 100.
Retina-lens preparations were post-fixed in cold 4% paraformaldehyde solution for 4 hours at 4°C.
The resulted retina-lens preparations were post-fixed in cold 4% paraformaldehyde solution for 4 hours at 4°C.
Breifly, the cells were rinsed with 1xPBS and fixed with cold 4% paraformaldehyde solution for 1 hour at 4°C.
For microtubule-associated protein (MAP-2) staining, the brains were fixed by immersion in a cold 4% paraformaldehyde solution in 0.1 M PB, pH 7.4.
At 24 h post injury, rats were anesthetized with an intraperitoneal injection of a ketamine:xylazine:acetylpromazine cocktail (50 10 1.67 mg/kg respectively) and perfused transcardially with saline followed by cold 4% paraformaldehyde solution in PBS for 30 min.
Cells were fixed with cold 2% paraformaldehyde solution for 15 min, washed in PBS 1X and then resuspended in PBS 1X supplemented with 0.25%BSA/0.01%% sodium azide containing an anti-Pgp polyclonal antibody [1 20 diluted, Mdr (H-241): sc-8313, Santa Cruz Biotechnology] or an isotype-matched negative control (DakoCytomation, Glostrup, Denmark) for 45 min at 4°C.
To evaluate the expression of HSP70 in treated lungs, lung tissue was fixed in cold 4% paraformaldehyde solution for 6 hours, dehydrated gradually with sucrose over a period of 24 hours, fixed in OCT compound (Tissue-Tek; Miles, Elkhart, IN, USA), and frozen.
To visualize native GFP or mRFP, we collected adult flies 2 11 days after eclosion, and brains were dissected in ice-cold 4% paraformaldehyde solution in PBS (1.86 mM NaH2PO4, 8.41 mM Na2HPO4, and 175 mM NaCl) and fixed for an additional 60 min at room temperature [ 19].
