Immunostaining primary antibody

Further dilute (if required) the resultant primary antibody Fab fragment complexes to optimal working concentration (usually about 1 5 pg/ml) in staining buffer containing 10% normal serum and then apply to the sample for 30 60 min at room temperature and proceed further with your standard immunostaining protocol. 

An additional control for the antibody specificity is the so-called absorption or preabsorption control, in which the primary antibody (prior to its use) is incubated for 1 h with a tenfold molar excess of the purified antigen. Absent or greatly diminished immunostaining should be obtained after application of this preabsorbed antibody. However, it is sometimes difficult to obtain the purified antigen therefore, it is rarely used routinely in immunohistochemical staining. Moreover, absorption of the antibody with the purified antigen does not always indicate that the antibody has bound to the same protein in the tissue (Burry 2000). 

A ready-to-use system for double HRP and AP immunostaining using a pair of primary antibodies, which come from mouse and rabbit, can be purchased from DCS Innovative Diagnostik Systeme (http //www.dcs-diagnostics.com/). 

The first immunostaining sequence blot excess blocking solution from sections, and incubate for 60 min at room temperature or overnight at +4°C with a correspondingly diluted first primary antibody. Wash sections in PBS or TBS for 3x3 min. 

Primary antibodies blot excess blocking solution from sections and incubate for 60 min at room temperature or overnight at +4°C with a mixture of correspondingly diluted unlabeled primary antibodies raised in two different host species (e.g., mouse and rabbit). When using fluorophore-labeled primary antibodies as in direct immunostaining method (one antibody layer), you may skip step (6) with secondary antibodies for indirect immunostaining method (two antibodies layers). Wash sections in PBS for 3 x 3 min. 

Basic protocol for multiple immunostaining using monoclonal primary antibodies of different IgG isotypes... 

The protocol for double/multiple immunolabeling using haptenylated primary antibodies is essentially the same as with primary antibodies of different IgG isotypes. These protocols can be easily customized depending on the availability of primary antibodies for your research requirements. For instance, you may have at your disposal a pair of monoclonal antibodies of the same IgG isotype, and only one of them is haptenylated. In this case, you have to carry out the immunostaining in two steps in the first step you visualize the unlabeled first primary antibody with a secondary species-specific antibody, and in the second step you can detect the second primary haptenylated antibody via another secondary antibody directed against the corresponding hapten. Should the hapten be a fluorophore, it can be visualized directly in a fluorescent microscope and you do not need the second step... 

Absorption or preabsorption control a control, in which the primary antibody (prior to its use) is incubated for 1 hr with a tenfold molar excess of the purified antigen. Absent or greatly diminished immunostaining should be obtained after application of this preabsorbed antibody. 

Direct and indirect immunostaining methods The direct method is a one-step staining method, and involves a labeled antibody reacting directly with the antigen in tissue sections. In this method, the primary antibody can be labeled with a fluorophore or biotin. In indirect immunostaining, the bound unlabeled primary antibody (first layer) is visualized with a secondary antibody (second layer) bearing label, such as a fluorophore, biotin or an enzyme. 

Appropriate controls should always be run with any immunocytochemical procedure. Controls may include omitting the primary antibody, substituting pre-immune serum, normal serum, or normal IgG for the primary antibody, adsorbing the primary antibody against the antigen, or immunostaining with an unrelated antibody. 

Once any necessary retitering of primary antibodies is accomplished, immunostaining results obtained with the automated stainer must be comparable to or better than those obtained with that laboratory s currently used manual methods. 

To assess any antibody adsorption to Leica manufactured vials, a primary antibody was stored in the reagent vials for up to 1 mo at 4°C. Subsequently, the antibody was removed, and the remaining reagents of an immunostaining run were sequentially added, as in the Cadenza vial and Code-On isolon experiments. Slight vial staining was seen after the 1-mo incubation period, indicating minimal antibody adsorption. 

FIGURE 12.3. Immunostained HER-2 protein in invasive breast carcinoma without antigen retrieval. Note homogeneous, predominantly membranous staining some cytoplasmic staining is also present. Monoclonal anti-c-erbB-2 antibody (diluted 1 200) (Triton, Almeda, CA) was used as the primary antibody. Courtesy of Jun Horiguchi. 

Immunoreactivity diminished or destroyed during dewaxing at high oven temperature. Oven temperature not to exceed 60 °C. NOTE The intensity of immunostaining may be diminished when tissue is exposed to prolonged heat. Refer to the primary antibody specification sheet for additional information. 29-33... 

---