Vector Laboratories

After washing 3x with PBS, place coverslips on slides with a drop of Vectashield mounting medium for fluorescence (Vector Laboratories, CA). 

For visualization of peroxidase label in tissue sections, Vector Laboratories offers the traditional substrates DAB and 3-amino-9-ethyl carbazole (AEC) as well as several proprietary substrates, producing colors as listed in Table 2.1. 

The alkaline phosphatase substrates form precipitates based on either reduction of tetrazolium salts or on the production of colored diazo compounds. Substrates Vector Red, Vector Black, Vector Blue, and BCIP/NBT available from Vector Laboratories produce reaction products which are red, black, blue and purple/blue... 

Table 2.1 Enzyme substrates from Vector Laboratories (http //www. vectorlabs.com/)...

Ready to use adhesive aqueous mounting media are available from Immunotech, Dako, Vector Laboratories, Molecular Probes, etc. 

Convenient ready-to-use nuclear counterstains in blue, green or red colour like Hematoxylin, Methyl Green and Nuclear Fast Red are available from Vector Laboratories (http //www.vectorlabs.com/), DAKO (http //www.dako.com/) and some other vendors, but if you are interested in preparing nuclear counterstains yourself, good and thorough procedures may be found in textbooks of Polak and Van Noorden (1997) and Renshaw (2007) or on the website http //www.ihcworld. com/counterstain solutions.htm. 

Based apparently on the same principle as described above, suitable but rather expensive kits exploiting monovalent Fab fragments to block endogenous tissue immunoglobulins have also been developed commercially, such as Mouse-on-Mouse (M.O.M. ) Kits by Vector Laboratories (http //www.vectorlabs.com/). Three Vector M.O.M. kits are available. These kits use the same blocking technology and biotin-avidin detection format, but offer a choice of using either an enzyme-based or fluorescent-based visualization method. 

Avidin-biotin complex horseradish peroxidase reagent (Vectastain ABC HRP Kit, Vector Laboratories, Inc., Burlingame, CA). 

Prepare the ABC reagent according to the manufacturer s instructions. For Vector Laboratories Vectastain Elite kit, add 2 drops of reagent A to 5 mL of PBS, then add 2 drops of reagent B. Mix and incubate for 30 min at room temperature. Additional volume may be prepared keeping the ratio of reagents constant. Repeat step 6. 

Avidin-biotin complex (Vector ABC Elite kit) (Vector Laboratories) ABC solution should be made fresh 10 min before use, so that complexes have time to form according to instractions of manufacturer. 

Endogenous biotin In certain tissues, most commonly kidney and liver, background staining can be high because of the presence of endogenous biotin. This can be minimized by preincubation of the sections with a dilute avidin solution, followed by incubation with a dilute biotin solution (Vector Laboratories, Burlingame, CA) before the application of the primary antibody. 

AP- and/or horseradish peroxidase (HRP)-conjugated antibodies for the haptens used for labeling probes (a direct detection method) or unconjugated antibodies for the haptens and AP- and/or HRP-labeled anti-species antibodies (an indirect detection method) (Vector Laboratories, Inc., Bnrlingame, CA, USA) (see Note 8). 

Vector Laboratories (Burlingame, CA, U.S.A.) Wak-Chemie (Bad Homburg, Germany)... 

The sections are incubated for 72 hr at 4°C with primary antiserum in PBS containing 2% normal serum and 0.25% gum arabic (Sigma). The sections are then incubated overnight at 4°C with a biotinylated secondary antibody (Vector Laboratories, Burlingame, CA), diluted 1 200. They are next incubated overnight at4°C with avidin-biotinylated peroxidase complex (ABC) diluted 1 100 in PBS containing 0.25% gum arabic. Note that the peroxidase-antiperoxidase procedure can also be used. 

Endogenous peroxidase activity is quenched by treatment with 0.5% H202 in methanol, immunostaining is carried out using a Vectastain Elite ABC kit, and visualization of the secondary antibody is achieved with DAB enhanced with nickel (Vector Laboratories). 

The cells and nuclei (0.5-1.0 X 106) are aliquoted into polystyrene tubes. Approximately 10 jjlI of normal horse serum (Vector Laboratories, Burlingame, CA) is added to block any nonspecific binding. The suspension is incubated with biotinylated antiestrogen monoclonal antibody (1D5, Dako Corp., Carpinteria, CA) at 1 25 dilution for 1 hr at 37°C. Aliquots are stained with 10 p,l of fluorescein isothiocyanate (FITC)-conjugated strepta-vidin (Dako buffer containing 0.1% Triton X-100). 

Avidin-biotin-PE complexes (Vectastain ABC-phycoerythrin, Vector Laboratories, Burlingame, CA, USA). Prepare according to the manufacturer s instrucdons that describe mixing a soludon of biodnylated PE with a soludon of avidin in the correct rado. 

Use, for example, Vectastain ABC method (Vector Laboratories, Burlingame, CA), or CSA Dakor (catalyzed signal amplification system for mouse primary antibodies) Dako and APAAP mouse or rat primary antibody kits (Dako). 

Dimyristoylphosphatidylethanolamine (DMPE), biotinyl-N-hydroxysuccinimide ester (BNHS), triethylamine, myoglobin, bovine serum albumin (BSA), lysozyme, guanidinium chloride, dimethylamino-cinnamaldehyde, acrylamide, ammonium persulfate, sodium dodecyl sulfate (SDS), and molybdenum blue reagent were all obtained from Sigma Chemicals and used without further purification as received. The compounds N, N, N, N -tetramethylethylenediamine (TEMED), N, N -methylene-bis-acrylamide, and Coomassie Brilliant Blue R-250 were from BioRad Laboratories. Avidin was obtained from Vector Laboratories with a quoted activity of 14 units/mg. All solvents were from Fisher Scientific. Water was passed through a Barnstead Nanopure system. 

Letter denotes vendor. A, Sigma Chemical Co. (St. Louis, MO) B, EY Laboratories, Inc. (San Mateo, CA) C, Vector Laboratories, Inc. (Burlingame, CA) D, Boehringer Mannheim Corp. (Indianapolis, IN). 

Working examples of practical algorithms used in the author s laboratory are presented in Chapter 12. The statistical data used to construct them were gathered over a period of several years using the following specimens that were fixed routinely in 10% neutral-buffered formalin primary antibody incubations at 4°C for 16 to 18 hours the Elite avidin-biotin-peroxidase complex method of immunodetection (Vector Laboratories, Burlingame, Calif) and the antibody reagents listed in Table 11.1. 

---