TITLE: Immunofluorescent labelling of paraffin brain tissue sections
AUTHORS: Birger Victor Dieriks, Helen Murray

[DESCRIPTION]
This immunofluorescence protocol permits the labelling of multiple antibodies on one paraffin tissue section. This protocol details the potential variations that detect the protein of interest using fluorescently labelled secondary antibodies.
 
Immunohistochemistry (IHC) combines anatomical, immunological and biochemical techniques to image discrete tissue components by using appropriately labelled antibodies to bind specifically to their target antigens in situ. IHC makes it possible to visualize and document the high-resolution distribution and localization of specific cellular components within cells and their proper histological context. There are multiple approaches and permutations in IHC methodology. 

 
Immunofluorescence permits labelling of multiple antibodies on one tissue section. For double or triple labelling, the primary antibodies must be raised in different species e.g. mouse, rabbit, chicken, guinea pig or goat. The corresponding secondary antibodies are conjugated to fluorophores with non-overlapping excitation/emission spectra (commonly used are fluorophores that excite at 488, 594 and 647nm). Antibodies from the same species but different Ig subtypes (e.g. mouse IgG1, IgG2a, IgG2b, IgG3, IgM) can be combined only if secondary antibodies that are highly specific to these subtypes are used. The specific excitation and emission wavelengths of different fluorophores permits labelling of multiple antibodies on one tissue section and visualisation of the labelling using widefield fluorescence or confocal microscopy. Hoechst or DAPI are commonly used nuclear counterstains for immunofluorescence.
 
Amplified IHC methods include fluorescent tyramide signal amplification (TSA, Figure 1e) and chromogenic 3,3′-Diaminobenzidine (DAB, Figure 1f) labelling. These methods detect the primary antibody using a secondary antibody conjugated to the horse-radish peroxidase (HRP) enzyme (Figure 2a). Alternatively, a biotin-conjugated secondary antibody can be applied, followed by a streptavidin conjugated HRP (Figure 2b). For chromogenic labelling, HRP converts the soluble substrate DAB into an insoluble brown product that can be visualised using brightfield microscopy. For fluorescence labelling, HRP converts an inactive tyramide-fluorophore complex into an active fluorescent complex that can be visualized using fluorescence microscopy.

 
Counterstains Nissl is a common nuclear counterstain for chromogenic labelling. Hoechst or DAPI are common fluorescent dyes used to stain DNA for fluorescent counterstaining.

[GUIDELINES]
Heat block reaches 60° Celcius and can cause burns when skin comes into contact with the hot surface.

[STEPS]
SECTION: Slide preparation
1. Label slides with a Statmark pen or a pencil.
 
Always include the proper controls for any fluorescent labelling. Control sections where the primary antibody was omitted should show no immunoreactivity.
SECTION: Dewaxing
3. Immediately following hotplate incubation, sections were submerged in 
30 min 2 x 100% xylene baths to remove paraffin wax. 
Subsequently, tissue sections were rehydrated by submerging slides in a series of alcohol baths 
15 min 2 x 100% EtOH 
5 min 95% EtOH 
5 min 80% EtOH 
5 min 75% EtOH 
5 min 3 x distilled H2O (dH2O) to wash off any remaining EtOH. 
  



2. Formalin-fixed paraffin-embedded tissue sections, when mounted on glass slides for microscopic examination, are placed on a hotplate with the tissue side up. This practice serves to

Facilitate Tissue Adherence: The hotplate softens the paraffin, allowing the tissue to flatten and firmly adhere to the glass slide. This ensures the tissue's integrity and correct orientation during staining and microscopic analysis.
Effectively Dehydrate: Paraffin sections often retain residual water. The hotplate aids in the removal of this water by evaporation, ensuring proper dehydration. Adequate dehydration is critical for subsequent staining and the preservation of tissue structures.
SECTION: Antigen retrieval in pressure cooker
4. Fill the pressure Cooker with approx. 600ml dH2O.

SECTION: Antigen retrieval in pressure cooker
4.2. Press start. 
This will initiate a heating protocol where the pressure cooker reaches 121 °C for 20 min, followed by an 100 min of gradual cool down. 


 
  



SECTION: Antigen retrieval in pressure cooker
4.1. Add slides in the cassette holder. Add the antigen retrieval buffer to the pressure cooker cartridge (Biovendor). Make sure to add sufficient antigen retrieval buffer to cover the tissue, but not to overfill
Frequently used buffers:
Sodium citrate buffer pH6
Tris-EDTA buffer pH9

 

  



SECTION: Antigen retrieval in pressure cooker
4.3. Pour out the antigen retrieval solution and replace it with 1x dH2O. Repeat 2x for about 5 min per wash. 
Use the pressure cooker cartridges for the wash steps in the rest of the protocol.
SECTION: Wax borders
7. Draw a hydrophobic barrier around each tissue section to reduce the reagents required.

This step can be done during the 3 x dH2O washes


 

SECTION: Permeablisation
8. Once wax borders are completed, replace dH2O with PBS-T and incubate for 15 min at 4 °C. 


 

SECTION: **OPTIONAL** TrueBlack autofluorescence quencher.
9. Trueblack treatment reduces the autofluorescence that is present in human brain sections. However, this additional step is only required when the signal-to-noise ratio of your labelling is low. TrueBlack can improve the signal for low-intensity labeling by reducing the background.
SECTION: Primary antibody incubation
11. For immunolabeling techniques like immunofluorescence, selecting the appropriate antibody is pivotal for the specificity and sensitivity of the assay. Antibodies serve as tools to specifically detect target antigens within a sample, and the precision of this detection largely hinges on the antibody's quality and specificity. The source of the antibody, marked by the supplier's reliability and reputation, plays a significant role since antibodies can differ substantially in their quality, purity, and overall performance based on their origin. Consequently, it is essential for researchers to meticulously assess their experimental needs and choose antibodies that align optimally with the requirements of their specific applications. This careful selection is key to ensuring the immunolabeling results are accurate.
SECTION: Permeablisation
8.1. Wash slides 3x 5 min in 1x PBS.

SECTION: **OPTIONAL** TrueBlack autofluorescence quencher.
9.1. Wash sections 3x 5 min in dH2O. PBS will cause the TrueBlack to clump on the tissue.
SECTION: **OPTIONAL** TrueBlack autofluorescence quencher.
9.2. Dilute TrueBlack (Biotum) reagent 1:20 in 70% EtOH (make 50µl per section depending on tissue size). Make sufficient true black solution to adequately cover the tissue section



SECTION: **OPTIONAL** TrueBlack autofluorescence quencher.
9.3. Place sections on a tray and add TrueBlack solution to sections for 30 s 
 

SECTION: **OPTIONAL** TrueBlack autofluorescence quencher.
9.4. Wash sections 3x 5 min in dH2O. PBS will cause the TrueBlack to clump on the tissue.
SECTION: Blocking for non specific labelling
10. In this step, we will block potential non-specific labelling. Ideally, this is performed using serum from animals in which the secondary antibodies were made (eg. if using goat secondary antibodies, then use goat serum; if using donkey secondary antibodies, then donkey serum). In practice, both can be used. HOWEVER, when a primary goat antibody is used, one cannot use goat serum in this protocol, as the secondary anti-goat antibody would label the entire section as it is covered with goat serum.  
SECTION: Blocking for non specific labelling
10.1. Make a humidified slide chamber by adding wet paper towels to one of the plastic slide chambers or trays. You can also use this time to check if you need to make a 10% blocking buffer and defrost the serum from  -20 °C freezer if needed.
  



SECTION: Blocking for non specific labelling
10.2. Make a 10% serum-blocking solution
500 µl serum stock solution + 4.5 ml 1x PBS. Use goat or donkey serum, depending on the species of your secondary antibodies. 10% serum solutions should be stored at 4 °C and discarded after two weeks. 10% serum solutions can be stored at -20 °Cand repeatedly freeze/thawed.
  

SECTION: Blocking for non specific labelling
10.3. Drain excess PBS from slides and move to the humid chamber. Add 100-200 µl of 10% blocking solution to each slide. Ensure the tissue is completely covered to the wax border. The volume of serum needed depends on the size of the tissue and doesn't have to be precise. Tissue needs to be entirely covered.
  

SECTION: Primary antibody incubation
11.1. Make immunobuffer: 1% normal goat (NGS) or donkey serum (NDS) in PBS
1 ml 10% NGS (or NDS) from step 6 + 9 ml 1x PBS. 1% serum solutions should be stored at 4 °C and discarded after two weeks. 1% serum solutions can be stored at -20 °Cand repeatedly freeze/thawed.
SECTION: Primary antibody incubation
11.2. Dilute antibodies in immunobuffer. 
Dilution factors depend on the company, individual antibody and region of interest. 
 
Thaw/store antibodies on ice during the protocol. Once thawed vortex and centrifuge before taking the required amount.
SECTION: Primary antibody incubation
11.3. Remove the 10% serum solution from the sections and return the slides to the humid chamber. No wash step is required here because the primary antibodies are diluted in the same serum.
SECTION: Primary antibody incubation
11.4. Add 100-200 µl of antibody solution to each slide. Volume can be optimised in the blocking step.
 

SECTION: Primary antibody incubation
11.5. Incubate at 4 °Covernight.

 

SECTION: Washing off primary antibody dilution
12. Antibody solution can be removed and discarded or recovered for additional antibody incubations. In our hands, we have successfully re-used primary antibody solutions up to three times. Store at -4 °C for up to two weeks or at -20 °C for long-term storage. 
SECTION: Washing off primary antibody dilution
12.1. Remove the primary antibody solution and transfer the slides back to the pressure cooker cartridge for 3 x 5 min washes with 1x PBS. 

SECTION: VARIATION** Block endogenous peroxidases.
13. This step is required for DAB labelling, Tyramide Signal Amplification (TSA) or any labelling that involves peroxidase-based signal amplification.

SECTION: VARIATION** Block endogenous peroxidases.
13.1. Make methanol blocking solution: 50% methanol + 1% H2O2  in MilliQ water

Incubate at Room temperature for 10 min

SECTION: Secondary antibody incubation
14. Secondary antibodies can be incubated 
overnight at 4 °C or 
at Room temperature for 180 min

SECTION: Secondary antibody incubation
14.1. Dilute secondary antibodies 1:500 in immunobuffer.  Add Hoechst diluted at 1:20,000 If doing fluorescence labelling. Hoechst counterstaining does not interact with secondary binding, so it is more efficient to include it in this step.
 

13.2. Wash 3x 5 min with 1x PBS and continue to the secondary antibody incubation step 
SECTION: Secondary antibody incubation
14.2. Transfer slides to the humid chamber and add ­­­­100-­200µl of secondary antibody solution to each slide
SECTION: Secondary antibody incubation
14.3. Incubate at Room temperature for 180 min. Cover the slide chamber with tinfoil to protect fluorophores from light.

SECTION: VARIATION** Tyramide amplification
15. We use Alexa Flour 488/594/647 Tyramide Superboost Kits (ThermoFisher) for primary antibody signal amplification. If additional secondary antibodies are simultaneously required, then these were diluted in 1x poly-HRP conjugated goat anti-mouse IgG secondary antibody (B40912-B, ThermoFisher) and added to the sections for 180 min at Room temperature as described above. 

 

Following incubation, the secondary antibody solutions were aspirated from slides and underwent 3 x 5 min PBS washes.


SECTION: VARIATION** Tyramide amplification
15.1. Prepare Tyramide working solution by diluting one drop of 100x hydrogen peroxide (H2O2) (B40912-
C2, ThermoFisher) in 1mL of MilliQ water for a 1:20 dilution. 
Dilute one drop of 20x Reaction buffer (B40912-C3, Invitrogen) in 1mL of MilliQ water. 
Add the diluted 100x hydrogen peroxide solution to the diluted 20x reaction buffer at 1:100 dilution.
SECTION: VARIATION** Tyramide amplification
15.2. Heat the combined solution  37 °C for  5 min
Add Alex Flour 488 Tyramide Reagent (B40912 Invitrogen) to the warm reaction buffer at a 1:100 dilution to produce a final Tyramide working solution.  
Make this solution fresh and use it immediately. 
SECTION: VARIATION** Tyramide amplification
15.3. Add 100-200 µl of the Tyramide working solution to each slide and incubate at 37 °C for 10 min 

SECTION: VARIATION** Tyramide amplification
15.4. Wash 3x 5 min with 1x PBS and continue to coverslipping.
SECTION: Coverslipping
16. Coverslip sections with Prolong gold.
 

SECTION: Coverslipping
16.1. Add a small amount of ProLong Gold (ThermoFisher) to the coverslip. 
Invert the glass slide so the tissue is between the coverslip and the glass slide. 
Gently push the glass slide on the coverslip, avoiding air bubbles and allowing the ProLong Gold to spread over the entire tissue section.

 

SECTION: **OPTIONAL** Formic Acid Antigen retrieval
5. Embedding tissue in paraffin often leads to the masking of antigenic sites due to the formation of cross-links between proteins, which can hinder the binding of antibodies to their target antigens. Formic acid, a strong acid, reverses this masking effect. It works by breaking down these protein cross-links, thus exposing the antigenic sites that were previously hidden. This additional formic acid antigen retrieval is especially beneficial for detecting certain types of antigens, such as prion proteins and other aggregated proteins, such as alpha-synuclein and Tau.
SECTION: **OPTIONAL** Formic Acid Antigen retrieval
5.1. Tap dry slides and add 99% formic acid for 4 min at Room temperature

 

SECTION: **OPTIONAL** Formic Acid Antigen retrieval
5.2. Wash immediately with dH20  3x  5 min per wash. 
SECTION: **OPTIONAL** Hydrochloric Acid Antigen retrieval
6. Hydrochloric Acid (HCl) antigen retrieval is particularly effective for visualizing nuclear antigens in paraffin-embedded tissue sections. By breaking down the protein cross-links that mask these antigens during formalin fixation, HCl enhances the exposure and binding of antibodies to nuclear epitopes, thus improving the clarity and specificity of immunohistochemical staining.
 

SECTION: **OPTIONAL** Hydrochloric Acid Antigen retrieval
6.1. Tap dry slides and add 2N HCL for 5 min at Room temperature .

 
  



SECTION: **OPTIONAL** Hydrochloric Acid Antigen retrieval
6.2. Wash immediately with dH20  3x  5 min per wash. 
SECTION: Antigen retrieval in pressure cooker
4.4.    if no additional antigen retrieval is required.

SECTION: **OPTIONAL** Formic Acid Antigen retrieval
5.3.    if no additional antigen retrieval is required.
SECTION: **OPTIONAL** Hydrochloric Acid Antigen retrieval
6.3.    if no additional antigen retrieval is required.
SECTION: Permeablisation
8.2.    if TrueBlack treatment is not required.
SECTION: Wax borders
7.1. Drain dH2O from the slides one slide at a time, but DO NOT let sections dry out. Draw a hydrophobic barrier around each tissue section using the ImmEdge pen (Vector Laboratories). To conserve reagents, draw barriers close to, but not on, sections. Tissue with a wax pen on top has high autofluorescence. 
Place the slide back into dH2O until all slides are done.

 

  



SECTION: Washing off primary antibody dilution
12.2.    if peroxidase blocking is not required.
SECTION: Coverslipping
16.3. Edges of cautofluorescenceoverslips can be sealed with nail polish.
 

SECTION: Storage
17. Store sections at 4 °C in slide boxes protected from light. 
 


2.1. Place all slides on a hot plate (Leica HI1220) at 60 °C for up to 60 min. 
 

  



SECTION: Coverslipping
16.2. An alternative method for coverslipping:
Gently push the coverslip on the glass slide, avoiding air bubbles and allowing the ProLong Gold to spread over the entire tissue section.

 

SECTION: **OPTIONAL** TrueBlack autofluorescence quencher.
9.5. Wash sections 3x 5 min in PBS.
2. 
2. 
2. 
2. 
SECTION: Secondary antibody incubation
14.4. Remove the secondary antibody solution.
Transfer the slides back to the pressure cooker cartridge for 3 x 5 minwashes with 1x PBS and continue to coverslipping