Immunofluorescence can also be used as a qualitative measure of protein expression.
Immunofluorescence protocol frozen section.
Immunocytochemistry and immunofluorescence protocol related fluorescence.
Direct vs indirect if.
Carry out incubations in a humidified chamber to avoid tissue drying out which will lead to non specific binding and high background staining.
Annexin v labeled with alexa fluor 488 in frozen rat placenta section by ihc immunohistochemistry.
Tissue preparation perfusion and fixation note.
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.
Cryosections adhered to slides from blocks embedded in oct using the 2 methylbutane isobutene method.
See cryoprotection and processing of embryonic tissue protocol.
Materials phosphate buffered saline pbs 1x paraformaldehyde pfa 4 see support protocol 1.
Cover sections with 4 formaldehyde diluted in warm 1x pbs.
Microscope slides pre coated.
Sections can be stored in a sealed slide box at 80 c for later use.
Dry the tissue sections overnight at room temperature.
Icc and if video protocol.
Store slides at 80 ºc until needed.
Modified from manipulating the mouse embryo 3.
For fresh unfixed frozen tissue fix immediately as follows.
Allow sections to fix for 15 min at room temperature.
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.
This portion of the protocol can be skipped if you are working with pre mounted tissue slides.
For fixed frozen tissue proceed with immunostaining section c.
This protocol is also suitable for 40µm free floating.
Section the frozen tissue block into a desired thickness typically 5 10 µm using the cryotome.
Brigitte arduini version 1 2015 mar 23.
Cut 4 8 um thick cryostat sections and mount on superfrost plus slides or gelatin coated slides.
Immunofluorescence on frozen sections.
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.
Store frozen blocks at 80 ºc.
Protocol for immunofluorescent staining of mouse frozen sections tissue.
Place the tissue sections onto glass slides suitable for immunohistochemistry e g.
The fluorescent immunohistochemistry immunofluorescence protocol below is intended for the fluorescent visualization of protein expression in frozen tissue sections.
