Preparation and Fluorescent IHC Staining of Frozen Tissue Sections

Fluorescent immunoﬂuorescence (IF) is a technique which utilizes ﬂuorescently labeled antibodies to visualize protein expression in a tissue section. Fluorescent visualization allows easier multiplex analysis of multiple proteins by immunohistochemistry (IHC) over traditional chromogenic detection system. The presentation below is intended for the ﬂuorescent visualization of protein expression in frozen tissue sections. It is just a general guide and staining conditions for specific antibody must be optimized according to different antigens of interest. Please read Protocol of Immunohistochemistry (IHC) in its entirety before beginning.

Contents

Step 1-1: Tissue Preparation-Perfusion and Fixation

Step 1-2: Tissue Preparation-Cryopreservation

Step 2: Blocking Non-specific Binding

Step 3: Antibody Staining

Step 4: Detection

Reagents

Coated slides (poly-L-Lysine)
4% paraformaldehyde
OCT cryostat sectioning medium
Incubation buffer
Blocking buffer
PBS
PBS-T: 0.4% Triton X-100 in PBS
Primary antibody
Secondary antibody

Methods

Note: Step 1 can be skipped if you are working with pre-mounted slides.

Step 1-1: Tissue Preparation-Perfusion and Fixation

Fix tissue by perfusing the animal with freshly prepared 4% paraformaldehyde or by immersing it in 4% paraformaldehyde for 4-24 hours at RT.

Note: Fixation time and temperature need to be optimized depending on the tissue type and size.

Cryoprotect the tissue by directly perfusing a sucrose solution and allow it to sink to the bottom of the vial.
Embed tissue in OCT cryostat sectioning medium and store at -80°C until ready for sectioning.

Note: Tissue can be safely stored for 6-12 months.

When ready for sectioning, move the embedded tissue directly into the cryostat and use OCT medium to mount it to the chuck. Allow the temperature of the tissue to equilibrate with the cryostat.
Cut the tissue in 5-20 µm thick sections. Mount tissue sections onto gelatin or poly-L-lysine coated slides by placing the cold sections onto warm slides.

Note: Slides can be safely stored for 6-12 months at -80°C until ready for staining.

Step 1-2: Tissue Preparation-Cryopreservation

After dissection, immediately snap freeze tissue with isopentane cooled by liquid nitrogen. After the tissue is frozen, place it in dry ice and move to -80°C until ready for cutting.
Embed tissue in OCT compound by slowly layering the compound so that the tissue does not thaw. Move the embedded tissue directly into the cryostat and use OCT medium to mount it to the chuck. Allow the temperature of the tissue to equilibrate with the cryostat.
Cut the tissue in 5-20 µm thick sections. Mount tissue sections onto gelatin or poly-L-lysine coated slides by placing the cold sections onto warm slides.

Note: Slides can be safely stored for 6-12 months at -80°C until ready for fixing. Uncut tissue can be restored at -80°C.

Remove slides from freezer and fix with cold fixative for 10 minutes. Proceed to staining.

Step 2: Blocking Non-specific Binding

Warm slides to RT. Wash slides twice with PBS.

Note: Fixation may result in epitope masking and non-specific background that can impact specific labeling. If necessary, a protocol for antigen retrieval can be performed at this time.

Draw a circle on the slide around the tissue with a hydrophobic barrier pen.
Wash the sections twice for 10 minutes with 1% animal serum in PBS-T.

Note: The species of the animal serum used is dependent on the host of your secondary antibody. For instance, when using a goat anti-mouse secondary, use goat serum.

Block non-specific binding by incubating the tissue sections with 5% serum in PBS-T for 30 minutes at RT.

Step 3: Antibody Staining

Add the primary antibody diluted in blocking buffer to the sections. Incubate at RT for 1-2 hours. Continue the incubation overnight at 4°C.

Note: The recommended starting dilution is 2-5 µg/ml if there are not specified antibody dilution.

Wash sections twice with 1% serum PBS-T for 10 minutes each.
Add a fluorescent label conjugated secondary antibody diluted in blocking buffer to the sections. Incubate at RT for 1-2 hours.
Wash sections twice with 1% serum PBS-T for 10 minutes each.

Step 4: Detection

Tap off excess wash. Apply one drop of ant-fade mounting medium to the slide.
Place a coverslip on the tissue sections. Circle the edges of the coverslip with clear fingernail polish to prevent the cells from drying. Allow the polish to air dry.
Slides may now be examined under a microscope with the appropriate ﬂuorescent filter sets. Be sure to limit slide exposure to light to prevent photobleaching.
Slides can be stored between -20°C and 4°C in a dark slide box or slide book.

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