Immunofluorescence Protocol Frozen Section

Microscope slides pre coated.

Immunofluorescence protocol frozen section.

Immunofluorescence on frozen sections. Dry the tissue sections overnight at room temperature. Allow sections to fix for 15 min at room temperature. Place the tissue sections onto glass slides suitable for immunohistochemistry e g.

Section the frozen tissue block into a desired thickness typically 5 10 µm using the cryotome. Cryosections adhered to slides from blocks embedded in oct using the 2 methylbutane isobutene method. Modified from manipulating the mouse embryo 3. Paraffin and frozen sections reagents can be applied manually by pipette or this protocol can be adapted for automated and semi automated systems if these are available.

Store frozen blocks at 80 ºc. Snap frozen fresh tissues in liquid nitrogen or isopentane pre cooled in liquid nitrogen embedded in oct compound in cryomolds. Immunofluorescence is commonly used to determine the cellular or tissue localization of a protein of interest. Materials phosphate buffered saline pbs 1x paraformaldehyde pfa 4 see support protocol 1.

For fixed frozen tissue proceed with immunostaining section c. Sections can be stored in a sealed slide box at 80 c for later use. See cryoprotection and processing of embryonic tissue protocol. Cut 4 8 um thick cryostat sections and mount on superfrost plus slides or gelatin coated slides.

The following immunohistochemistry ihc protocol has been developed and optimized by r d systems ihc icc laboratory for fluorescent ihc experiments using frozen tissue samples. Direct vs indirect if. The fluorescent immunohistochemistry immunofluorescence protocol below is intended for the fluorescent visualization of protein expression in frozen tissue sections. Immunofluorescence can also be used as a qualitative measure of protein expression.

Cover sections with 4 formaldehyde diluted in warm 1x pbs. Carry out incubations in a humidified chamber to avoid tissue drying out which will lead to non specific binding and high background staining. Protocol for immunofluorescent staining of mouse frozen sections tissue. This protocol is also suitable for 40µm free floating.

Annexin v labeled with alexa fluor 488 in frozen rat placenta section by ihc immunohistochemistry. Immunofluorescence on frozen tissue sections bio protocol. This portion of the protocol can be skipped if you are working with pre mounted tissue slides. For fresh unfixed frozen tissue fix immediately as follows.

Immunocytochemistry and immunofluorescence protocol related fluorescence. Tissue preparation perfusion and fixation note. Store slides at 80 ºc until needed. Nagy gertsenstein vintersten and behringer ed.