Rabbit Anti-MCM2 Proliferation Pairs Staining Procedure

Paraffin section preparation

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.

Frozen section preparation

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. 

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.

Protein blocking

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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Addition of primary antibodies

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.


Addition of secondary antibodies

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.

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Chromogenic development

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.

Fluorescence development

Fluorescent slides should be mounted in anti-fade medium with a counterstain such as DAPI. Slides should be stored in the dark.

Top tips

  1. Titrate antibodies individually for optimal staining before combining in dual staining.
  2. For chromogenic staining, we recommend using an HRP/DAB system for MCM2 detection, as the dark brown stain gives the best visualization after the addition of a blue hematoxylin nuclear counterstain.
  3. Careful differentiation of the hematoxylin stain in 1%HCL/70% aqueous ethanol will produce the optimum level of nuclear counterstain that won’t mask fainter MCM2 staining.
  4. Use of an additional nuclear antigen should be avoided for chromogenic staining, as interpretation of co-staining levels will be difficult.
  5. Ensure that secondary antibodies are raised in appropriate species e.g. using a goat anti-rabbit IgG:HRP with a rabbit anti-mouse IgG:AP will result in dual labeling of the mouse primary antibody.
  6. Use species cross-adsorbed secondary antibodies to prevent direct interaction with the tissue section or other secondary antibodies.

Recommended secondary antibodies

The following secondary antibodies are recommended for use with these primary antibodies.

Table 1. Secondary Antibodies.

Primary Antibody

Chromogenic Detection

Fluorescent Detection

MCM2 Rabbit IgG

644004 (Alk Phos), STAR54 (HRP)

STAR36D488, STAR36D549

Cell Specific Antibody Mouse IgG

STAR117A (Alk Phos), STAR117P (HRP)

STAR117D488, STAR117D549

Cell Specific Antibody Rat IgG

STAR131A (Alk Phos), STAR72 (HRP)

STAR71D549

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