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