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NDT401-bc Tissue preparation & immunofluorescence labeling with 2 primary antibodies on free-floating sections

This service includes tissue preparation, sectioning, immunostaining, mounting, coverslipping and labeling the slides. As a result, you will receive up to 60 immunostained per brain or per tissue block ready for microscopic observations. To express our appreciation, a set of Nissl (or H & E) stained sections adjacent to those used for immunostaining will also be provided for you without additional cost.

Procedure: Following cryoprotection, tissue will be rapidly frozen in isopentane pre-cooled to -70ºC. The frozen tissue will then be cut on a cryostat and collected in our unique section cryoprotection solution (cf. Product NDT301). Subsequently, the sections cut from various levels (or the levels of your choice) will be processed free-floating for immunostaining with 1 specific antibody according to immunogold labeling technique1 (cf. photo samples below).

Left: Cofocal image of BrdU-immunoreactivity. 30 £gm cryostat section was cut from the hippocampal dentate gyrus of a mouse that survived for 24 hr after the injection with 5-bromo-2-deoxyuridine (BrdU). This section was processed free-floating according to the indirect fluorescence method.

Middel: Confocal image of NeuN-immunoreactivity.  The same section as shown on the left was processed free-floating for NeuN-immunoreactivity according to the indirect fluorescence method. Note NeuN-labeled granule cells in the dentate gyrus. Right: Colocalization of BrdU- and NeuN-immunoreactivities. A digital overlay of the 2 images shown on the left. Note that the regions of colocalization, reflecting the additive effect of superimposed green and red pixels, appear in yellow.

1. A quotation is required before placing an order.
2. The investigator needs to provide unfixed frozen tissue.
3. Please contact us for more information.

1. Coons, A.H. (1958) Fluorescent antibody methods. In J.F. Danielli (ed): General Cytochemical Methods. New York: Academic Press, pp. 399-422.

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