For fresh unfixed frozen tissue fix immediately as follows.
Immunofluorescence protocol frozen section.
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.
Annexin v labeled with alexa fluor 488 in frozen rat placenta section by ihc immunohistochemistry.
This ihc protocol provides a basic guide for the fixation cryostat sectioning and staining of frozen tissue samples.
Snap frozen fresh tissues in liquid nitrogen or isopentane pre cooled in liquid nitrogen embedded in oct compound in cryomolds.
For fixed frozen tissue proceed with immunostaining section c.
Carry out incubations in a humidified chamber to avoid tissue drying out which will lead to non specific binding and high background staining.
Immunofluorescence can also be used as a qualitative measure of protein expression.
Direct vs indirect if.
Place the tissue sections onto glass slides suitable for immunohistochemistry e g.
Store slides at 80 ºc until needed.
Cryosections adhered to slides from blocks embedded in oct using the 2 methylbutane isobutene method.
Nagy gertsenstein vintersten and behringer ed.
See cryoprotection and processing of embryonic tissue protocol.
The fluorescent immunohistochemistry immunofluorescence protocol below is intended for the fluorescent visualization of protein expression in frozen tissue sections.
Cut 4 8 um thick cryostat sections and mount on superfrost plus slides or gelatin coated slides.
Dry the tissue sections overnight at room temperature.
Protocol for immunofluorescent staining of mouse frozen sections tissue.
Section the frozen tissue block into a desired thickness typically 5 10 µm using the cryotome.
Icc and if video protocol.
Immunofluorescence on frozen sections.
Cover sections with 4 formaldehyde diluted in warm 1x pbs.
Materials phosphate buffered saline pbs 1x paraformaldehyde pfa 4 see support protocol 1.
Tissue preparation perfusion and fixation note.
Immunocytochemistry and immunofluorescence protocol related fluorescence.
Modified from manipulating the mouse embryo 3.
This portion of the protocol can be skipped if you are working with pre mounted tissue slides.
Sections can be stored in a sealed slide box at 80 c for later use.
Microscope slides pre coated.
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.
Brigitte arduini version 1 2015 mar 23.
Immunofluorescence on frozen tissue sections bio protocol.
Allow sections to fix for 15 min at room temperature.
