Appendix Multiple Antigen Labeling General Notes Immunofluorescence Staining Methods Discovery Through Color Appendix Appendix 1: Counterstain/Substrate Compatibility Table This table is designed as a reference to determine the optimal counterstain/substrate combination for your application. Vector® Hematoxylin & Hematoxylin QS H-3401, H-3404 Vector® Methyl Green H-3402 Vector® Nuclear Fast Red H-3403 Substrate Catalog No. DAB (brown) DAB-Ni (gray/black) AEC (red) TMB (blue) VECTOR® VIP (purple) VECTOR® SG (blue/gray) VECTOR® NovaREDTM (red) SK-4100 SK-4100 SK-4200 SK-4400 SK-4600 SK-4700 SK-4800 Excellent Contrast Excellent Contrast Excellent Contrast Color Incompatibility Fair Contrast Poor Contrast Excellent Contrast Excellent Contrast Fair Contrast* Counterstain Incompatibility** Counterstain Incompatibility Excellent Contrast Good Contrast Excellent Contrast*** Fair Contrast Good Contrast Color Incompatibility Excellent Contrast Poor Contrast Excellent Contrast Color Incompatibility VECTOR® RED (red) VECTOR® BLACK (brown/black) VECTOR® BLUE (blue) BCIP/NBT (blue/violet) SK-5100 SK-5200 SK-5300 SK-5400 Excellent Contrast Excellent Contrast Color Incompatibility Color Incompatibility Excellent Contrast Excellent Contrast* Good Contrast Excellent Contrast* Color Incompatibility Excellent Contrast Excellent Contrast Excellent Contrast GLUCOSE OXIDASE NBT (purple/blue) GLUCOSE OXIDASE TNBT (black) GLUCOSE OXIDASE INT (red/purple) SK-3100 SK-3200 SK-3300 Color Incompatibility Excellent Contrast Good Contrast Excellent Contrast Excellent Contrast Counterstain Incompatibility** ExcellentContrast Excellent Contrast Fair Contrast * This substrate shows a slight decrease in sensitivity following the Methyl Green protocol. This decrease can be minimized by reducing the heat incubation and acetone rinse times in the Methyl Green protocol. ** Substrate dissolves in acetone wash. ***A slight color change in Vector® NovaREDTM reaction product may be seen using Methyl Green. Counterstains should be optimized for each tissue type, antigen unmasking protocol, and immunocytochemical staining intensity desired. Appendix 2: Quenching Endogenous Enzyme Activity Quenching Endogenous Peroxidase Activity Method 1. 3% H2O2 in water. Incubate for 5 minutes. Rinse with water for 2-3 minutes. This is the most rapid and simplest technique for quenching, however the bubbling that might occur may damage the morphology of frozen sections and specimens with large amounts of endogenous enzyme activity (e.g., blood smears, etc.). This is a good general block. Method 2. 0.3% H2O2 in methanol. Incubate for 20-30 minutes. Rinse with water for 2-3 minutes. This is the method of choice for frozen sections and specimens with large amounts of endogenous enzyme activity (blood smears, cytospins, etc.). The concentration of H2O2 can be doubled and/or incubation time shortened as appropriate for the specimen. Methanol accelerates the destruction of the heme groups so a lower concentration of H2O2 can be used for a longer period of time. This is also a good general block except for cell surface markers. Method 3. 0.180 g β-D(+) glucose, 5 mg glucose oxidase, 6.5 mg sodium azide in 50 ml PBS. Incubate sections for 1 hour at 37 oC. Rinse in PBS 3 x 5 minutes. This reaction slowly and steadily produces very low concentrations of H2O2 by enzymatic reaction. This method con- sistently and completely inhibits peroxidase activity. (Andrew S.M., Jasani, B.; Histochem J. 19, 426- 430,1987.) Quenching Endogenous Alkaline Phosphatase Activity Method 1. If the endogenous activity is an isoenzyme other than the intestinal form, it can be inhibited by the addition of levamisole (Cat. No. SP-5000) to the buffer used to prepare the substrate solution. Levamisole is a competitive inhibitor of most alkaline phosphatase (AP) activity in tissues but is not bound by the iso- form of AP used in detection systems. Intestinal alkaline phosphatase can be inhibited by either of the following two methods: Method 2. Prior to staining, treat the sections with 20% acetic acid at 4 °C for 15 minutes. Method 3. Treat the sections with 2.3% periodic acid for 5 minutes and 0.02% potassium boro- hydride for 2 minutes. (Bulman A.S. and Heyderman E.; J. Clin. Pathol. 34, 1349-1351, 1981). Appendix 3: Buffer Recipes for Substrate Solutions PBS (10 mM phosphate, 150 mM NaCI, pH 7.5) 1.42 g Na2HPO4 8.75 g NaCI to 950 ml distilled water pH to 7.5 with phosphoric acid, bring up to final volume of 1 liter 100 mM Tris-HCI, pH 8.2 or 9.5 12.11 g Tris Base to 950 ml distilled water pH to 8.2 or 9.5 with HCI, bring up to final vol- ume of 1 liter NOTE: For 200 mM Tris-HCI, double the quanti- ty of Tris Base.