Protein A gold-silver staining method for light microscopic immunohistochemistry.

Abstract

Summary.A The immunohistochemical methods employing colloidal gold-labeled immunoglobulins are subject to the serious limitation that immunoglobulins of all animal species will not necessarily bind firmly to colloidal gold. To circumvent such a limitation, an indirect immunohistochemical method named the protein A gold technique has been developed, in which a protein A-colloidal gold complex was used as the second stepreagent (ROMANO and ROMANO,1977; ROTH and BINDER, 1978; BENDAYAN et al., 1980; ROTH et al., 1981a, b; RoTH,1982). The protein A gold technique for immunohistology was originally developed for electron microscopy, and RoTH (1982) was the first to successfully employ this technique for light microscopy, demonstrating reddish reaction products in the rat endocrine pancreas.In the present study, attempts have been made to establish an improved method for the protein A gold technique for light microscopy, in which the contrast of the immunoreactive sites obtained by the protein A gold technique are intensified by means of our improved procedure of physical development.The method presented here has been tentatively named the protein A gold-silver staining. MATERIALS AND METHODSPieces of tissues from the splenic portion of the adult rat pancreas were fixed in Bouin's fluid for 18 hr, dehydrated in a graded ethanol series, cleared in xylene and embedded in paraffin. Sections were cut at a thickness of 4 pm, deparaffinized, hydrated and subjected to the following staining procedures. The sections were: 1) immersed for 15 min in three changes of 0.01 M phosphate buffered saline (PBS) (pH 7.4); 2) treated for 60 min with 1 % ovalbumin in PBS; 3) incubated for 60 min with guinea pig anti-porcine insulin serum (DAKO corporation, USA) diluted 1:400 with 1 °o bovine serum albumin in 0.01 M phosphate buffered saline (BSA-PBS) at room temperature in a moist chamber; 4) rinsed in four changes of PBS for 30 min; 5) treated for 15 min with loo ovalbumin in PBS; 6) incubated for 30 min with protein A gold complex preparation (E-Y Lab. Inc., USA., particle size, approximately 10 nm) diluted 40 times with BSA-PBS; 7) rinsed in four changes of 0.01 M phosphate buffer (pH 7.4) for 30 min; 8) physically developed with a solution of the formula noted below for 45-50 min at 20°C in a dark room; 9) washed in running tap water for 5 min and then immersed for 1 min in a photographic fixer diluted 5 times with water; 10) rinsed in running tap water for 10 min; 11) briefly counterstained with 0.1°o nuclear fast red in 5°o alminium sulfate aqueous solution; 12) dehydrated, cleared and mounted in a Bioleit.The formula of the developing solution used at step (8) is as follows : Solution A : 20% gum arabic aqueous solution, 45 ml; 10 silver nitrate aqueous solution, 1 ml. The gum arabic solution was prepared by centrifugation at 18,000 rpm for 30 min at 0°C and separation of the supernatant for use. Solution B comprised: distilled water, 15 ml; bromohydroquinone, 200 mg; citric acid, 300 mg. The working developing solution was prepa...

Enhanced expression of endothelin B receptor at protein and gene levels in human cirrhotic liver

“…21 In control liver, ultrastructural localization of ETAR was hardly detectable on HSCs and SECs, while ETBR was evident on HSCs as well SECs. In cirrhotic liver, ETBRs were strongly expressed on HSCs.…”

“…The working developing solution was prepared by mixing solution A and B in a dark room under illumination of a photographic safety lamp. 21 For electron microscopy, the tissue specimens were treated with PBS for 15 minutes three times, fixed in 1.2% glutaraldehyde buffered with 0.01% phosphate buffer (pH 7.4) for 1 hour at 4°C, treated with graded series of ethanol solutions, and post-fixed with 1% osmium tetroxide in 0.01% phosphate buffer (pH 7.4). The liver tissues were embedded in Epon (Polyscience, Inc. Warrington, PA).…”

Expression of adhesion molecules on mature cholangiocytes in canal of Hering and bile ductules in wedge biopsy samples of primary biliary cirrhosis

“…Solution B contained 200 mg of hydroquinone (Kanto Chemical Co., Tokyo, Japan) and 300 mg of citric acid monohydrate (Kanto Chemical Co., Tokyo, Japan) in 10 mL of distilled water. The working developing solution was prepared by mixing solutions A and B in a dark room under illumination of a photographic safety lamp [24] . For electron microscopy, the tissue specimens processed for light microscopy as above were treated thrice, with PBS for 15 min, and fixed for 1 h at 4 in 1.2% glutaraldehyde buffered with 0.01% phosphate buffer (pH 7.4), followed by a graded series of ethanol solutions.…”

A Double Protein A-gold-silver Staining Method Using One Step Development for Light Microscopy.

“…In light microscopic immunohistochemistry, a series of immunogold-silver staining methods have been established, such as the IgG-gold-silver (IGGS) [2, 3,[9][10][11]18,20,21] and protein A-gold-silver (PAGS) [4,6,7,13,14] techniques.…”

“…In the PAGS staining method [4][5][6] using silver nitrate as the developer [6], reaction products first appear in pale brown shades and then gradually change their colors from light brown to dark black during the course of development.…”