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Tris/EDTA/Tween pH9 is recommended for HIER for maximum sensitivity, although Citrate pH 6 can be used as an alternative. For chromogenic staining, endogenous peroxidase activity should be blocked with BUF017B while endogenous alkaline phosphatase activity can be blocked using levamisole during the alkaline phosphatase detection step.
Note: if an alkaline phosphatase conjugate is being used for detection, all wash and incubation steps should be performed in Tris buffered saline instead of PBS.
Acetone is recommended for fixation, although staining has been achieved using formaldehyde as a fixative. In either case, we recommend treatment of fixed frozen sections with 0.1% Triton X100 for 5 min to enable full access to the nuclear epitope for anti-MCM2. For chromogenic staining, endogenous peroxidase activity should be blocked with 0.3% hydrogen peroxide/0.09% sodium azide/PBS for 30 min, while endogenous alkaline phosphatase activity can be blocked using levamisole during the alkaline phosphatase detection step.
Non-specific protein interactions should be blocked by incubation of the sections with 10% FCS or 1% BSA for 30 min.
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Rabbit anti-MCM2 concentration should be titrated by the user, as staining intensity will be influenced by the nature of the section, species and detection method. Generally a concentration between 0.5 and 10 µg/ml will produce good staining intensity. The concentration of the second counterstaining primary should be based on the manufacturer’s recommendation or the user’s predetermined value. Primary antibodies may be added simultaneously and incubated for 30 min to one hr at RT or overnight at 4°C if preferred. After incubation, the slides should be rinsed for 3 times (3x) 3 min with PBS or TBS as appropriate.
Fluorescent dye or enzyme conjugated secondary antibodies should be used at the manufacturer’s recommended dilution, and can be added simultaneously. The incubation should typically last for 30 min to 1 hr at RT, and in the dark for fluorescent conjugates. After incubation, the slides should be rinsed for 3 times (3x) 3 min with PBS or TBS as appropriate.
Chromogenic enzyme substrates must be added sequentially due to variations in reaction conditions. We recommend using the alkaline phosphatase substrate first, using the manufacturer’s recommended protocol with the inclusion of levamisole. After color development, the slide should be washed before the addition of the peroxidase substrate. The slide should again be washed after color development and counterstained using hematoxylin or a suitable alternative before mounting.
Fluorescent slides should be mounted in anti-fade medium with a counterstain such as DAPI. Slides should be stored in the dark.
The following secondary antibodies are recommended for use with these primary antibodies.
Table 1. Secondary Antibodies.
MCM2 Rabbit IgG
644004 (Alk Phos), STAR54 (HRP)
Cell Specific Antibody Mouse IgG
STAR117A (Alk Phos), STAR117P (HRP)
Cell Specific Antibody Rat IgG
STAR131A (Alk Phos), STAR72 (HRP)