Immunofluorescence Protocol Frozen Section

Protocol for immunofluorescent staining of mouse frozen sections tissue.

Immunofluorescence protocol frozen section.

Immunofluorescence is commonly used to determine the cellular or tissue localization of a protein of interest. Cryosections adhered to slides from blocks embedded in oct using the 2 methylbutane isobutene method. Immunofluorescence on frozen tissue sections bio protocol. Nagy gertsenstein vintersten and behringer ed.

Allow sections to fix for 15 min at room temperature. Snap frozen fresh tissues in liquid nitrogen or isopentane pre cooled in liquid nitrogen embedded in oct compound in cryomolds. This protocol is also suitable for 40µm free floating. This ihc protocol provides a basic guide for the fixation cryostat sectioning and staining of frozen tissue samples.

Modified from manipulating the mouse embryo 3. Materials phosphate buffered saline pbs 1x paraformaldehyde pfa 4 see support protocol 1. For fresh unfixed frozen tissue fix immediately as follows. Immunocytochemistry and immunofluorescence protocol related fluorescence.

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. Icc and if video protocol. Direct vs indirect if. Place the tissue sections onto glass slides suitable for immunohistochemistry e g.

Cut 4 8 um thick cryostat sections and mount on superfrost plus slides or gelatin coated slides. This portion of the protocol can be skipped if you are working with pre mounted tissue slides. Store frozen blocks at 80 ºc. For fixed frozen tissue proceed with immunostaining section c.

See cryoprotection and processing of embryonic tissue protocol. Store slides at 80 ºc until needed. Immunofluorescence on frozen sections. Microscope slides pre coated.

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. Sections can be stored in a sealed slide box at 80 c for later use. Tissue preparation perfusion and fixation note. Carry out incubations in a humidified chamber to avoid tissue drying out which will lead to non specific binding and high background staining.

Brigitte arduini version 1 2015 mar 23. Dry the tissue sections overnight at room temperature. 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.