Immunofluorescence Protocol Frozen Section

Tissue preparation perfusion and fixation note.

Immunofluorescence protocol frozen section.

Modified from manipulating the mouse embryo 3. Icc and if video protocol. Immunofluorescence is commonly used to determine the cellular or tissue localization of a protein of interest. Immunocytochemistry and immunofluorescence protocol related fluorescence.

Brigitte arduini version 1 2015 mar 23. Immunofluorescence on frozen tissue sections bio protocol. Dry the tissue sections overnight at room temperature. Place the tissue sections onto glass slides suitable for immunohistochemistry e g.

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. Immunofluorescence can also be used as a qualitative measure of protein expression. Nagy gertsenstein vintersten and behringer ed.

Annexin v labeled with alexa fluor 488 in frozen rat placenta section by ihc immunohistochemistry. For fixed frozen tissue proceed with immunostaining section c. 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. 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.

Store frozen blocks at 80 ºc. The fluorescent immunohistochemistry immunofluorescence protocol below is intended for the fluorescent visualization of protein expression in frozen tissue sections. Cryosections adhered to slides from blocks embedded in oct using the 2 methylbutane isobutene method. Sections can be stored in a sealed slide box at 80 c for later use.

Section the frozen tissue block into a desired thickness typically 5 10 µm using the cryotome. Cover sections with 4 formaldehyde diluted in warm 1x pbs. Cut 4 8 um thick cryostat sections and mount on superfrost plus slides or gelatin coated slides. Allow sections to fix for 15 min at room temperature.

This ihc protocol provides a basic guide for the fixation cryostat sectioning and staining of frozen tissue samples. Immunofluorescence on frozen sections. Snap frozen fresh tissues in liquid nitrogen or isopentane pre cooled in liquid nitrogen embedded in oct compound in cryomolds. Protocol for immunofluorescent staining of mouse frozen sections tissue.

Direct vs indirect if. See cryoprotection and processing of embryonic tissue protocol. Store slides at 80 ºc until needed. Carry out incubations in a humidified chamber to avoid tissue drying out which will lead to non specific binding and high background staining.