INSITU BLOTTING - A NOVEL METHOD FOR DIRECT TRANSFER OF NATIVE PROTEINS FROM SECTIONED TISSUE TO BLOTTING MEMBRANE - PROCEDURE AND SOME APPLICATIONS

We describe a novel technique for direct transfer of native proteins f rom unfixed frozen tissue sections to an immobilizing matrix, e.g., ni trocellulose, polyvinyliden difluoride, or positively charged nylon me mbranes. Proteins are directly blotted onto the membrane, providing op timal accessibility for molecular detection but retaining the anatomic localization at the cellular level. Within 10 min a maximum protein t ransfer is achieved independent of the protein molecular weight. The t otal protein bound was 80% of the maximal binding capacity of the blot ting membrane and independent of the section thickness. These results indicate that the proteins that bind to the membrane originate from th e cut cell monolayer that has direct contact with the blotting membran e. This in situ blotting method provides direct protein mapping from a single cell layer of a tissue section. The procedure includes cryosec tioning at 20 mum and collecting sections on a dry blotting membrane a t -20-degrees-C. For protein transfer the blotted sections are thawed and incubated for 10 min with Tris buffer. After incubation the sectio ns are removed from the membrane by high-pressure spray. The blotted m embranes can be subjected to several detection assays. In the present study the presence of several proteins was demonstrated in brain and t hymus by immunochemical and enzyme histochemical procedures.