TITLE: Multiplex Immunofluorescence on Fresh Frozen Tissue
AUTHORS: Maya Brewer, Yuantee Zhu, Elizabeth Neumann, Danielle Gutierrez, Jeff Spraggins, Mark de Caestecker

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Scope:</div><div class = "text-block">To describe the procedure for multiple cycles of immunofluorescence on human kidney tissue embedded in carboxymethylcellulose.</div><div class = "text-block">Expected Outcome:</div><div class = "text-block">Kidney tissue sections that have been tagged with antibodies for imaging microscopy.</div></div>

[STEPS]
?. [Immunofluorescence]
If sections are frozen, allow them to equilibrate to room temperature (~15 minutes). Place slides in a humidified chamber. They will remain in the chamber throughout this protocol.
?. [Immunofluorescence]
While slide is dry, using a hydrophobic pen, draw a large barrier around the section. Do not allow pen to touch the section.
?. [Immunofluorescence]
Post-fix sections in 10% formalin for 5 minutes.
?. [Immunofluorescence]
Remove fixative by tilting the slide, allowing the solution to flow from the section into the humidified chamber.
?. [Immunofluorescence]
Wash sections in 1X PBS for 5 minutes three times. For this, tip solution off the slide into the chamber, add PBS to the slide using a pipettor, tip again and repeat.
?. [Immunofluorescence]
Incubate sections for 5 minutes with 50 mM glycine (dilute stock in 1X PBS). This reduces autofluorescence by reducing free aldehyde groups.
?. [Immunofluorescence]
Remove glycine, and wash sections in PBS for 5 minutes twice.
?. [Immunofluorescence]
Incubate sections with 3% hydrogen peroxide (dilute 30% H2O2 in 1X PBS) at room temperature for 10 minutes to further reduce autofluorescence. This reduces non-specific fluorescence signals
?. [Immunofluorescence]
Remove hydrogen peroxide, and wash sections in PBS for 5 minutes four times.
?. [Immunofluorescence]
Block sections for 60 minutes with 1X Universal Blocking Reagent (UBR) at room temperature.
?. [Immunofluorescence]
Dilute 10X blocking reagent to 1X using 9-parts ddH2O to 1-part UBR.
?. [Immunofluorescence]
Dilute primary antibody to desired working concentration in Antibody Diluent Reagent during blocking step.
?. [Immunofluorescence]
Add diluted antibody to section and incubate overnight at 4˚C.
?. [Immunofluorescence]
Remove solution, and wash sections with PBS for 5 minutes twice.
?. [Immunofluorescence]
If primary antibody is directly conjugated with a fluorophore, skip to #16.
?. [Immunofluorescence]
If using indirect immunofluorescence, dilute fluorophore-conjugated secondary antibody using Antibody Diluent Reagent.
?. [Immunofluorescence]
Add antibody solution to sections and incubate for 60 minutes at room temperature.
?. [Immunofluorescence]
Remove solution, and wash sections with PBS for 5 minutes twice.
?. [Immunofluorescence]
Incubate sections in Hoechst 33342 (1:10,000 dilution of 20mM solution in 1X PBS) for 10 minutes.
?. [Immunofluorescence]
Remove Hoechst 33342, and wash sections with PBS for 5 minutes twice.
?. [Immunofluorescence]
Mount slides in 70% glycerol in PBS. Do not seal the coverslips as they will need to be removed later.
?. [Immunofluorescence]
Because slides are not sealed, they must be kept horizontal to prevent the coverslip from falling off, and in the humidified chamber to keep them from drying out.
?. [Immunofluorescence]
Image
?. [Immunofluorescence]
Store slides at 4˚C in a moisture chamber.
?. [De-Coverslipping]
Remove coverslip from sections by incubating slides in a vertical staining jar filled with PBS for 15 minutes with slight agitation (i.e. plate rocker).
?. [De-Coverslipping]
Slowly lift slide from vertical jar and allow coverslip to release from slide via gravity.
?. [De-Coverslipping]
Wash slide in PBS for 5 minutes three times to remove any residual glycerol.
?. [De-Coverslipping]
Place slides back in vertical staining jar full of PBS with slight agitation to wash.
?. [Fluorophore I]
Make a solution of 4.5% hydrogen peroxide and 24 mM sodium hydroxide made up in PBS.
?. [Fluorophore I]
Add bleaching solution to each section and incubate at room temperature for 90-120 minutes in the presence of white light (LED light).  
?. [Fluorophore I]
For this, place slides on a plastic surface on top of the LED light. We often use multiple plates for this.
?. [Fluorophore I]
Halfway through this incubation, the solution may be removed by pipette and replaced with fresh solution to ensure complete inactivation occurs from the LED light.
?. [Fluorophore I]
Remove solution and wash sections in PBS for 5 minutes four times.
?. [Fluorophore I]
After inactivating the fluorophores, slides are mounted in 70% glycerol and imaged to confirm complete fluorophore inactivation, followed by the removal of the coverslip, and three 5 minutes washes in PBS. Hoechst stain will not bleach and is necessary for image registration later.
?. [Subsequent Immunofluorescence Cycles]
Dilute fluorophore-conjugated primary antibodies using Antibody Diluent Reagent.
?. [Subsequent Immunofluorescence Cycles]
Add diluted antibodies to each section and incubate overnight at 4˚C.
?. [Subsequent Immunofluorescence Cycles]
Remove antibody solution and wash sections with PBS for 5 minutes twice.
?. [Subsequent Immunofluorescence Cycles]
Incubate sections in Hoechst (1:10,000 dilution in 1X PBS) for 5 minutes (this may not be necessary, but does no harm).
?. [Subsequent Immunofluorescence Cycles]
Remove Hoechst, and wash sections with PBS for 5 minutes twice.
?. [Subsequent Immunofluorescence Cycles]
Mount slides in 70% glycerol in PBS.
?. [Subsequent Immunofluorescence Cycles]
Image
?. [Subsequent Immunofluorescence Cycles]
Store slides at 4˚C in a moisture chamber.
?. Sections II-IV are repeated for each remaining cycle.