NOTE: If possible, carry out the following steps with floating sections and on a rotary shaker.

1. Use some method to remove autofluorescence.
2. Incubate with the permeabilization solution (0.1% Triton X-100 in PBS) for 15 minutes at room temperature (RT).
3. Incubate with blocking solution (1% FCS + 0.1% Triton X-100 in PBS) for 1-2 hours at RT.
4. Incubate with the primary antibody diluted in the blocking solution, overnight at 4°C. Centrifuge it previously at 12,000xg in the minifuge for 10 minutes at 4°C. For sections larger than 50 microns it is possible to extend the incubation for two nights by doing it at RT. In this case, add 0.02% Sodium Azide.
5. Wash 5 times for 5 minutes with PBS.
6. Incubate with the secondary antibody diluted in the blocking solution for 12-18 hours at RT in the dark. Centrifuge previously at 12,000xg in the minifuge for 10 minutes at 4°C.
7. Wash with the permeabilization solution for 1 hour with frequent changes.
8. LWash with PBS for another hour with frequent changes. The sections can be stored in PBS or 4% Paraformaldehyde (PFA) for several days. It is preferable not to go to the next step until they are going to be observed.
9. Put the sections on slides, add mounting medium and place a cover on top.

NOTE: the whole process is carried out with the tissue sections (50-100 µm) in suspension.

1. Fix in PFA 4% overnight (ON) at 4°C.
2. Wash 2 times for 5 minutes with PBS pH 7.4.
3. Incubate with the permeabilization solution (0.1% Triton X-100 in PBS) for 15 minutes.
4. Incubate with 0.003% H₂O₂ in PBS for 30 minutes at room temperature (RT) to reduce endogenous peroxidase activity.
5. Wash with PBS 3 times for 5 minutes.
6. Incubate with blocking solution (1% serum + 0.1% Triton X-100 in PBS) for 15 minutes at room temperature.
7. Incubate with the primary antibody overnight at 4°C diluted in the blocking solution. Centrifuge it previously for 10 minutes at 12,000xg in the minifuge at 4°C.
8. Wash 5 times for 5 minutes with PBS.
9. Incubate with the peroxidase-coupled secondary antibody for 1h at RT diluted in the permeabilization solution. Centrifuge it previously for 10 minutes at 12,000xg in the minifuge at 4°C.
10. Wash 5 times for 5 minutes with PBS.
11. Wash 2 times for 5 minutes with 100 mM Tris pH 7.6.
12. Develop the reaction with 0.5-1 mg/mL DAB (3,3'-diaminobenzidine) + 0.06% H₂O₂ in 100 mM Tris pH 7.6. No more than 10-15 minutes.
13. The reaction can be intensified with NiCl2 and CoCl₂ (1%) in 100 mM Tris pH 7.6.
14. Stop the reaction by adding 0.05% Azide.
15. Wash 3 times for 5 minutes with PBS.
16. Mount the sections with mounting medium on a slide. Place a coverslip on top.

Protocol elaborated from “Unraveling human adult hippocampal neurogenesis”. Nat Protoc 15, 668–693 (2020).

NOTE: The following conditions are suitable for particular primary antibodies and 50 µm human brain sections. Try different incubation times to set the antibodies of interest.

1. Fixation: Immediately after extracting and dissecting the tissue, immerse it in 4% PFA for 24h at 4 ° C. Wash 3 times for 30 seconds with 0.1N PB (phosphate buffer) at room temperature (RT).
2. Embed the tissue in agarose:
   • Prepare a solution of 10% sucrose-4% low-melting point agarose, heated in the microwave to make it liquid and fill the well of a M6-M12 plate.
   • Put the plate on ice and immerse the tissue. It may float for the first 2 minutes. Push it to the bottom to avoid it and wait for the agarose to completely solidify.
   • Cut a cube containing the tissue, with 2-3 mm extra agarose in all dimensions.
3. Sectioning: Cut 50 µm sections on the vibratome, collecting the sections in 0.1 N PB.
4. Incubate the sections with 0.5% NaBH4 in 0.1N PB for 30 minutes (this step is not necessary if the tissue comes from mice). Put the plate at RT on an orbital shaker with gentle shaking. The appearance of bubbles is frequent.
5. Wash 5 times for 30 seconds with PB-Triton-BSA (PBT-BSA).
6. Antigen retrieval (antigen retrieval) with citrate (HC-AR), if necessary:
   • Fill 10 mL glass vials with 5 mL of 1x Citrate buffer (pH 6.0) preheated at 90 ° C (make the buffer just before using).
   • Place 1 to 4 sections belonging to the same type of sample in each glass vial, and close without sealing it to avoid a pressure increase during microwave heating.
   • Subject the vials to 5-6 cycles of 10-20 seconds in a microwave (800W at maximum power). To avoid tissue damage, the liquid should never be boiled, in which case microwave cycles should be suspended. Wait a few seconds between each microwave cycle.
     
     NOTE. This process should be repeated until the edges of the sections begin to curve slightly, stopping as soon as this effect occurs. Excessive folding of the edges of the sections should be avoided to avoid damaging the sample, the appearance of backgrounds and to preserve the specificity of the signal.
     
     
   • Close the vials well and immerse them in a water bath at 80 ° C for 20 minutes.
   • Leave the vials closed for 20 minutes at RT.
   • Remove the sections from the vials with the help of a soft brush and wash them 5 times for 30 seconds in 0.1 N PB.
7. Incubate with the primary antibody. Put the primary antibody diluted in PBT-BSA in each well of an M24 plate, place the sections in the wells and make sure that they are floating (350-400 µl per well in this type of plate is suitable for incubating up to two sections). Incubate for 5 days at 4 ° C with gentle shaking. Incubation conditions and time must be adjusted experimentally for the antibodies used.
8. Wash sections 5 times for 30 seconds with PBT-BSA.
9. Incubation with the secondary antibody. Put the secondary antibody diluted in PBT-BSA in each well of an M24 plate (500 µl per well is suitable for up to three sections). Incubate in the dark overnight (ON) at 4 ° C with gentle shaking.
   
   IMPORTANT. From this step the sections should be kept in the dark. For this, aluminium foil can be used.
   
   
10. Wash the sections three times for 30 seconds with 0.1 N PB.
11. Incubation with DAPI (optional). To stain the nuclei, the floating sections can be stained with DAPI (1: 5000 v/v in 0.1 N PB) for 10 min at RT with gentle shaking. A volume of 500 µl per well (M24) can be used to stain a maximum of three sections.
12. Wash sections 3 times for 30 seconds with 0.1 N PB at RT.
13. Autofluorescence removal:
    • Immerse the sections in 1 mL of 70% ethanol for 5 minutes with gentle shaking. In an M24 a volume of 500 µl is sufficient for a maximum of three sections per well.
    • Incubate sections in commercial Autofluorescence Eliminator Reagent (EMD Millipore, Cat#2160) (Sudan Black diluted in ethanol) for 5 minutes with vigorous shaking to prevent reagent precipitation. In an M24 a volume of 200-300 µl should be sufficient (take in mind that the sections will be stained black). This treatment should be adjusted according to the native autofluorescence of the tissue.
    • Wash the sections 3 times for 1 minute with 1 mL of 70% EtOH with strong shaking. Make sure that the Autofluorescence Eliminator precipitates are removed with these washes.
14. Wash the sections 3 times for 30 seconds in 1 mL of 0.1 N PB at RT with gentle agitation.
15. Mounting the sections. Place the sections on a slide treated with 2% gelatine. The use of a commercial mounting medium or not is optional. Once the mounting medium has been added, place a coverslip and allow the preparation to dry, protecting it from light. Wipe off excess mounting medium carefully.
16. Samples can be stored at RT in the dark. It is recommended to acquire the images within the month following assembly.

USED SOLUTIONS

• 10% sacarose-4% low-melting agarose (500 mL)
  • Heat 500 mL of 0.1 N PB.
  • Add 50 g of sucrose and mix on a magnetic stirrer.
  • Once the sucrose has completely dissolved, add 20 g of Low Melting Point agarose. Warm in a microwave until bubbles begin to form. Shake and rewarm. Repeat until transparent and slimy.
    
    NOTE. The solution can be stored at -20 ° C for 6 months or at 4 ° C for a week.
    
    
• 0.1 N PB Solution (1 L)
  • Add 14 g of K₂HPO₄ to 800 mL of bidistilled water.
  • Once completely dissolved, add 2.65 g of NaH₂PO₄-H₂O.
  • Adjust the pH to 7.4 and make up to 1L with distilled water.
    
    NOTE. It can be prepared and stored at RT for up to 6 months.
    
    
• PB-Triton-BSA Solution (PBT-BSA) (100 mL)
  • Dissolve 1 g of BSA (1% w/v) in 100mL of 0.1 N PB.
  • Add 1 mL of Triton-X-100 (1% v/v) and mix on a magnetic mixer until completely dissolved.
    
    NOTE. The PBT-BSA solution can be stored at 4°C for one week
    
    
• 2% gelatine-coated glass slides
  • Dissolve 6 g of porcine gelatine in 300mL of hot bidistilled water on a magnetic mixer.
  • When completely dissolved, add 0.5 g of KCr(SO₄)₂.
  • Pour 250 mL of this solution into the slide cell.
  • Place 10 slides in the holder and immerse them in the gelatine solution for 1 min.
  • Allow slides to dry ON before use.
    
    NOTE. These slides can be stored at RT for 6 months.

1. Fix with 4% PFA for 20 minutes.
2. Block for 1 h in PBT 10 (10% BSA + 0.1% Tween-20 in PBS).
3. Incubate with primary antibody: dilute in PBT 1 (1% BSA + 0.1% Tween-20 in PBS) and incubate overnight (ON) at room temperature (RT).
4. Wash several times in PBT 1 (1% BSA + 0.1% Tween-20 in PBS) for 1-2 hours.
5. Incubate with secondary antibody: dilute in PBT 0.1 (0.1% BSA + 0.1% Tween-20 in PBS) and incubate 2-4 hours in the dark.
6. Nuclei staining (optional): incubate with Hoescht 5 µg/ml in PBT (0.1% Tween-20 in PBS) for 10 minutes.
7. Wash several times with PBT (0.1% Tween-20 in PBS) for 30 minutes.
8. Mount with Vectashield.

• Various markers (Enzo Life Sciences y Thermofisher).
• Cell membrane markers (CellVue).
• Expression vectoirs and fluorescent proteins (EvroGen).
• SNAP-tag and CLIP-tag (New England Biolabs).
• SiR dyes: cytoskeleton in vivo markers (Spirochrome).

• Component to add to the culture medium and maintain the pH (Hepes).
• Media to avoid “bleaching” (antifading ProLong ^TM Live and Live Cell Visualization Medium DMEM ^GFP_-2).
• Specific culture media for in vivo experiments (Live Cell Imaging Solution and LiveLight Photostable Media).
• To decrease autofluorescence in cultures (BackDrop™)

• Gels to create cameras for the study of aquatic microorganisms (Carolina Observation Gel).
• Culture and hybridization chambers (Incubations chambers and Specimen Preparation and Embedding Supplies).
• To maintain humidity (with gas exchange) in long-term experiments (Anti-Evaporation Oil , Mineral oil and FoilCover).
• Perfusion systems available at the SMOC.