Immunostaining unlabeled

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. 

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... 

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. 

Fig. 9.2 A-C Adjacent sections of field CA1, from a rat 4 weeks post-kainate injection. A is a section stained with Perl s stain, showing an increase in the number of ferric iron positive cells (arrows) in the degenerating CA field (see Fig. 9.1 A). B is a section stained with Turnbull s blue stain, showing an increase in ferrous iron positive cells (arrows) in the degenerating CA field (see Fig. 9.IB). C is a ferritin immunostained section, showing an increased number of ferritin positive cells (arrows). D-F adjacent sections of field CA1, from a rat 8 weeks post-kainate injection. D is a section stained with Perl s stain, showing an increase in the number of ferric iron positive cells, compared to 4 weeks post-injection (see A). E is a section stained with Turnbull s blue, showing an increase in the number of ferrous iron-containing cells, compared to 4 weeks post-injection (see B). F is a ferritin-stained section, showing a decrease in the number of ferritin positive cells (arrows) compared to 4 weeks post-injection (see C). The spherical, heavily ferric or ferrous iron-laden cells shown in D and E are unlabeled for ferritin. Scale=50 /xm. Reproduced with kind permission from Huang and Ong (2005) Experimental Brain Research 161 502-511. Springer.

Fig. 9. Aspartate aminotransferase immunoreactivity in glutamic acid decarboxylase (GAD)-immunoreactive neuronal processes in the cerebral cortex. sAAT but not mAAT is colocalized with GAD in fine, probably axonal processes (arrows). Rat sections were double-immunostained by incubation with a mixture of anti-sAAT or mAAT rabbit serum and anti-GAD sheep serum, then with biotinylated anti-rabbit IgG donkey antibody, and finally with Texas Red-conjugated avidin and fluorescein-labeled anti-sheep IgG donkey antibody. The photographs in each row were taken at the same site under different excitations. Asterisks in (a) and (o ) indicate the unlabeled cell body of a pyramidal neuron.

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