Fluorophore-conjugated secondary antibodies used in this study were from Jackson ImmunoResearch Laboratories (West Grove, PA) and included Cy3-conjugated AffiniPure donkey anti-rat IgG (cat. #712-165-153), Cy3-conjugated AffiniPure donkey anti-mouse IgG (cat. #715165-150), and fluorescein (DTAF)-conjugated AffiniPure donkey anti-rabbit IgG (cat. #711-095-152), each at a dilution of 1:100 in antibody diluent. For further amplification, secondary antisera used were biotin-SP-conjugated AffiniPure F (ab’)2 fragment donkey anti-mouse IgG (H&L) (cat. #715-066-151) or biota-SP-conjugated AffiniPure F (ab’)2 fragment donkey anti-rabbit IgG (cat. #711-066-152), in conjunction with Cy3-conjugated strep-tavidin (cat. #016-160-084) or fluorescein (FITC)-conju-gated streptavidin (cat. #016-090-084). buy cheap antibiotics
Cryosections were dried at room temperature under vacuum (30 min) prior to fixation in acetone (5 min). After washing in hypertonic PBS solution (hPBS; 274 mM NaCl. 5.37 mM KCl, 8.10 mM Na2HPO4, 1.47 mM KH2PO4, pH 7.2; three times, 5 min each), some sections were denatured with acidified urea solution (6 M urea, 0.1 M glycine, 0.1 M HCl, pH 3.5) for 30 min and then washed again (three times, 5 min each in hPBS). The denaturation step was omitted when the mouse monoclonal antibodies were used. Nonspecific staining was inhibited by the preincubation of sections with 10% normal donkey serum in hPBS (30 min). Sections were incubated overnight with either specific primary antiserum or nonimmune control serum.