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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.