Mayer’s hematoxylin

Stain in Mayer s hematoxylin solution for 30 sec 10 min (usually 1 min). Ready-to-use Mayer s hematoxylin solution with stabilizers purchasable from Sigma (Cat. No. MHS-16) can be stored for years without losing its potency. 

Counterstain with Mayer s hematoxylin 1-5 min depending on the concentration and color intensity desired (see Note 9). 

Mayer s hematoxylin. Use for counterstaining tissue sections after completing BISH assays. 

Soak the slide in a Coplin jar of Mayer s hematoxylin solution for 5 s at room temperature. 

The sections are incubated in the rabbit polyclonal anti-VEGF (Santa Biotechnology, CA) and diluted 1 100 for 30 min at room temperature. Antigen-antibody reaction is detected using the biotin-streptavidin-based detection kit (Dako). The reaction is developed using DAB+hydrogen peroxide and counterstained with Mayer s hematoxylin. Exclusion of the primary antibody serves as a negative control. The results of this procedure are shown in Fig. 1.7. 

A cocktail (1 1) of two monoclonal anticyclin Dl/bc 1-1 antibodies were used P2D11F11 (diluted 1 40) and 5D4 (diluted 1 100) these two antibodies can be obtained from Vecta Laboratories, Inc., Burlingame, CA, and Immunotech, Westbrook, ME, respectively. The avidin-biotin immunoperoxidase detection system employing DAB is used as the chromogen (Ventana Biotek). After counterstaining in dilute Mayer s hematoxylin, the sections are dehydrated and mounted in Permount. 

Figure 7.2 A. Immunostaining of caspase 14 in exponentially growing NHEKs (400x). B. NHEKs treated with 50 pM EGCG for 24 h (400x). C. Confluent OSC2 cells (400x). Pre-immune rabbit serum was used as negative control (data not shown), and cell nuclei were counterstained with Mayer s hematoxylin.

AEC is alcohol soluble and its crisp red color contrasts well against hematoxylin. To avoid removal of the alcohol-soluble product, a non-alcohol based stain like Mayer s hematoxylin should be used. AEC has two reactive sites so that when one is converted it turns red but when both are saturated a green-brown color results. Aquamount causes slow loss of the stain and glycerol mounting is required, rendered permanent by sealing the edges of the cover slip. AEC may be chosen because it may be a lower-risk carcinogen compared to DAB. 

The washed filter is then stained with Mayer s hematoxylin, cleared with propanol, subsequently treated with xylene. 

To prevent the silver intensificadon from fading, fix the reaction product with 5% sodium thiosulfate for 5 min and wash with water. Counterstain as required (1 min with Mayer s hematoxylin). 

Fig. 28-4. Nasal tissue, BALB/c mouse, 2 days after exposure to aerosolized VEE virus. Note immunoreactive olfactory epithelium and olfactory nerves. Alkaline phosphatase-labeled streptavidin method using rabbit antiserum to VEE virus (Mayer s hematoxylin counterstain, original magnification x 300).

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