Protocol for immunofluorescent staining of mouse frozen sections tissue.
Immunofluorescence protocol frozen section.
Cover sections with 4 formaldehyde diluted in warm 1x pbs.
Materials phosphate buffered saline pbs 1x paraformaldehyde pfa 4 see support protocol 1.
Snap frozen fresh tissues in liquid nitrogen or isopentane pre cooled in liquid nitrogen embedded in oct compound in cryomolds.
Immunofluorescence on frozen tissue sections bio protocol.
Carry out incubations in a humidified chamber to avoid tissue drying out which will lead to non specific binding and high background staining.
Immunocytochemistry and immunofluorescence protocol related fluorescence.
Store frozen blocks at 80 ºc.
Immunofluorescence on frozen sections.
Dry the tissue sections overnight at room temperature.
Place the tissue sections onto glass slides suitable for immunohistochemistry e g.
Store slides at 80 ºc until needed.
This protocol is also suitable for 40µm free floating.
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.
Brigitte arduini version 1 2015 mar 23.
This ihc protocol provides a basic guide for the fixation cryostat sectioning and staining of frozen tissue samples.
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.
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.
Allow sections to fix for 15 min at room temperature.
Modified from manipulating the mouse embryo 3.
Annexin v labeled with alexa fluor 488 in frozen rat placenta section by ihc immunohistochemistry.
See cryoprotection and processing of embryonic tissue protocol.
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.
Microscope slides pre coated.
Tissue preparation perfusion and fixation note.
Nagy gertsenstein vintersten and behringer ed.
Sections can be stored in a sealed slide box at 80 c for later use.
For fresh unfixed frozen tissue fix immediately as follows.
Icc and if video protocol.
Cut 4 8 um thick cryostat sections and mount on superfrost plus slides or gelatin coated slides.
Section the frozen tissue block into a desired thickness typically 5 10 µm using the cryotome.
