Distribution of the «1 to «6 Chains of Type IV Collagen: MATERIALS AND METHODS(4)

MATERIALS AND METHODS(4)

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

Immunohistochemistry

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.

This entry was posted in Follicles and tagged Bovine, Chains, Collagen, Follicles.