Immunohistochemistry Techniques to Analyze Cellular Proliferation and Neurogenesis in Rats Using the Thymidine Analog BrdU

All procedures follow the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications N°. 8023, revised in 1978) and local Mexican laws to minimize the number of animals used and their suffering. The Ethics Committee of the Universidad Iberoamericana approved the experimental protocols for using animals in this study.

1. Reagent preparation and setup

NOTE: Most of the solutions can be prepared days before use unless specified otherwise.

1. BrdU solution
   1. Retrieve the BrdU solution from -20 °C freezer and allow it to equilibrate at room temperature (RT).
   2. Calculate the mass of BrdU needed for a dose of 50 mg/kg according to the body weight of the rat. Calculate volume of 0.9% saline solution (0.9 g NaCl in 100 mL of sterile H₂O) needed for a working solution of 20 mg/mL. Prepare an excess to provide at least 0.5 mL per rat per injection.
      NOTE: The dose administered to experimental animals should be safe, with minimal side effects, and effective. It has been reported that duration of staining with 100 mg/kg BrdU does not outweigh the potentially higher toxicity compared with the 50 mg/kg dose⁷. No significant differences were found in the number of BrdU-labeled cells/mm³ for 50 and 100 mg/kg i.p. in rats⁷. It is preferable to inject a small dose to minimize the suffering of the animals.
   3. Weigh out BrdU solution and add it to the saline solution in a conical tube and vortex.
      NOTE: Pre-heat the saline solution at 45‒50 °C in a water bath for volumes bigger than 1 mL.
   4. Place the tube in a water bath at 50 °C for 10‒15 min and vortex every 2‒3 min until completely dissolved. Filter the solution with a syringe filter for sterile injection. Cover the tube with tin foil, cool it down at room temperature and use immediately.
      CAUTION: BrdU solution is toxic and potentially carcinogenic. Prepare it in the fume hood. BrdU solution must be handled with proper protective equipment (PPE). It is recommended to prepare the solution immediately before use. However, the solution is stable for 24 h under RT. Please protect it from light.
2. To prepare 1 L of 0.1 M phosphate buffered saline (PBS) at pH 7.4, add 240 mg of potassium phosphate monobasic (KH₂PO₄), 1.44 g of sodium phosphate dibasic (Na₂HPO₄), 200 mg of potassium chloride (KCl), and 8 g of sodium chloride (NaCl) to 800 mL of double distilled water (ddH₂O) under constant stirring. Adjust the pH to 7.4 and add double distilled H₂O up to total volume of 1 L. Store at 4 °C for up to 1 week.
3. For 100 mL of PBS+, add 3% (3 mL) of normal horse serum and 0.3% (300 µL) of Triton X-100 to 0.1 M PBS (pH 7.4). Store in 20‒50 mL aliquots at -20 °C for up to 3 months.
   NOTE: Alternatively, TBS can be used instead of PBS. Any other serum different from the host’s antibodies and experimental tissue is suitable.
4. For 100 mL of PBS++, add 10% (10 mL) of normal horse serum and 0.3% (300 µL) of Triton X-100 to 0.1 M PBS pH 7.4. Store in 20‒50 mL aliquots at -20 °C for up to 3 months.
5. For 1 L of cryoprotectant solution, mix 250 mL of ethylene glycol and 250 mL of glycerol, constantly stir until mixed. Slowly bring to 1 L with PBS. Filter with grade 4 (20‒25 μm) filter paper. Store at 4 °C or RT for up to 1 year.
6. Prepare 4% paraformaldehyde in 0.1 M PBS (PFA solution) as follows. For 1 L of solution, add 40 g of paraformaldehyde powder slowly to 800 mL of 60‒65 °C 0.1 M PBS under constant stirring. Stir until paraformaldehyde is completely dissolved while controlling the temperature (60‒65 °C). If necessary, add a few drops of 1 M NaOH to clarify the solution. When the solution reaches room temperature, filter with grade 4 (20-25 μm) filter paper.
   CAUTION: Paraformaldehyde is toxic and is suspected of being a carcinogen, prepare in the fume hood. Store at 4 °C for and preferably use within to 2 days. PFA ready-to-use solution is commercially available.
7. For 1 L of 10 mM Sodium citrate buffer (SCB) at pH 6, add 1.204 g of sodium citrate (dihydrate), and 1.134 g of citric acid to 800 mL of double distilled H₂O under constant stirring. Adjust the pH to 6.0 and add ddH₂O up to 1 L. Store at 4 °C for up to 6 months.
8. Prepare 50 mL of 2 N HCl by slowly adding 8.25 mL of 12 N HCl (concentrated stock solution) to 41.75 mL of ddH₂O under constant stirring.
   CAUTION: Prepare in the fume hood. Solution must be prepared immediately before use.
   NOTE: 2 N HCl will be used for DNA denaturation, a crucial step. As BrdU is incorporated into the DNA, HCl is used to open the DNA bonds allowing BrdU antibody access BrdU within the DNA.
9. Prepare endogenous peroxidase blocking solution as follows. Prepare 100 mL of 0.6% hydrogen peroxide by mixing 2 mL of 30% hydrogen peroxide with 98 mL of ddH₂O under constant stirring.
   NOTE: The solution must be prepared immediately before use. Keep it in the dark as H₂O₂ is light sensitive. PBS or TBS can be used instead of water.
10. Prepare avidin-biotin complex (ABC) solution as per the instructions from the manufacturer. For 5 mL of ABC in 0.1 M PBS, add 2 drops (≈100 µL) of reagent A and mix, and then add 2 drops (≈100 µL) of reagent B and mix.
    NOTE: Solution must be prepared and allowed to tumble-roll for 20‒30 min before use.
11. Prepare DAB (Diaminobenzidine) Peroxidase (HRP) substrate using the kit by following the instructions of the manufacturer. To 5 mL of ddH₂O, add 2 drops (≈ 84 µL) of reagent 1 and mix, add 4 drops (≈ 100 µL) of reagent 2 and mix, then add 2 drops (≈ 80 µL) of reagent 3 and mix. Finally, if desired, add 2 drops (≈ 80 µL) of reagent 4 (Nickel) and mix.
    NOTE: Solution must be prepared immediately before use.
    CAUTION: DAB is toxic and potentially carcinogenic. It must be handled with care and discarded as per the hazardous waste regulation at each institution. To inactivate DAB, add several drops of bleach (sodium hypochlorite); the solution will turn black.
12. Prepare 100 mL of cresyl violet solution by adding 100 mg of cresyl violet acetate and 250 μL of acetic acid to 80 mL of ddH₂O at 55‒60 °C. Adjust the volume to 100 mL, filter, and store at 4 °C in a dark-colored vessel.
    NOTE: The user is encouraged to test different concentrations of the cresyl violet solution before using it on valuable tissue samples. The result may be darker for counterstaining with some tissue samples, which may decrease the ability to count BrdU positive cells accurately.

2. Thymidine analog BrdU administration

1. Restrain the experimental animal (e.g., 90-day-old male Wistar rat weighing 350 g), by immobilizing the lower abdominal cavity.
2. Administer the BrdU solution (50 mg/kg) intraperitoneally (i.p.) using a 23 G needle and 1 mL syringe.
   NOTE: Adjust the injection volume as per weight of the animal. Use a 23‒27 G needle and a 1‒5 mL syringe for adult rats. The maximum tolerable intraperitoneal injection volume in the adult rat is 10 mL. Different routes can be used to administer the BrdU solution¹⁴. For example, intraperitoneal injection or oral administration through drinking water.

3. Tissue preparation

NOTE: Three-month-old rats were allowed ad libitum access to physical activity (endless wheel) for seven days. On the day 6, rats were injected with BrdU (step 2) 3 times at intervals of 12 h. Perform steps in section 3 after 8 h from the last BrdU injection.

1. Inject pentobarbital (50 mg/kg i.p.) and wait a few minutes until the animal is deeply anesthetized.
   NOTE: Make sure that the animal is completely anesthetized before continuing. Carefully pinch one of the legs or the tail. If the animal reacts to the stimulus, wait a few more minutes. If the animal does not react to the pinch, go to the next step.
2. Expose the heart by cutting the abdominal cavity skin below the breastbone, taking apart the ribs, and cutting the diaphragm.
3. Transcardial perfusion fixation
   1. Insert a needle into the left ventricle and make a small incision in the right atrium. Using a pump or gravity, perfuse (flow rate 5‒7 mL/min.) 0.1 M PBS until entire blood is drained out, and the solution becomes clear.
   2. Using a pump or gravity, perform cold perfusion (flow rate 5‒7 mL/min) with PFA solution to fix the tissue until the tail becomes rigid.
      NOTE: Usually, a 300 g rat requires around 100‒150 mL of the PFA solution. The tissue fixation is optional. Thereby, the brain can be extracted for use in various processes to minimize animal usage in the experiments.
4. Dissection and post-fixation
   1. Decapitate the animal, and gently extract the brain from the skull. Immerse the brain into a conical tube containing PFA solution (~40 mL for a 250 mg rat) for 1‒2 days at 4 °C.
      NOTE: Do not over-fixate (more than 48 h), because this can deplete the tissue staining due to the unavailability of antigens.
   2. Prepare 100 mL of 30% sucrose solution, adding 30 g of sucrose to 70 mL of 0.1 M PBS solution under constant stirring. Add 0.1 M PBS solution to 100 mL. Immerse the brain into a conical tube with a 30% sucrose solution (35 mL) for approximately 1‒2 days at 4 °C until the brain sinks to the bottom of the tube.
5. Cutting coronal brain sections
   NOTE: Using a cryostat-microtome requires guidance and training. For detailed instructions, see reference¹⁵.
   1. Submerge the whole brain into iso-pentane at -80 °C and keep it at -80 °C for 10 min. Place the brain in an embedding matrix on a cryostat-microtome plate.
      NOTE: Under certain conditions, rapid freezing of the brain at -80 °C can cause fracture or damage to the tissue. The user should be aware of this problem. If this is the case, use -20 °C iso-pentane to freeze the brain.
   2. Using a cryostat-microtome (temperature at -25 to -20 °C) cut coronal sections of 40 μm thickness. Sequentially transfer sections into a 24-well cell culture plate with cryoprotection solution following the guide in Figure 2. Store at -20 °C until use, for up to few months.
      NOTE: Hereafter, process all tissue in free-floating serial sections of 40 µm in 12-well plates with mesh inserts in gentle and continuous agitation (10 rpm). It is possible to store brain sections for years under the right conditions.

Figure 2: Schematic illustration of sequentially transferring sections from cryostat-microtome into a 24-well cell culture plate with cryoprotection solution. Begin at A1-well and place the next slices into row A; after A6-well move to the next row B, so on. When arriving D6, go back to A1 and continue. This arrangement allows Nth (e.g., sixth for neurogenesis, equivalent to the content of one column) section quantification of an entire brain region. Please click here to view a larger version of this figure.

4. Immunostaining

NOTE: See Table 1 for the summary of the advantages and disadvantages of each technique.

1. Detection of BrdU using peroxidase reaction with DAB
   NOTE: Perform steps 4.1.1 to 4.1.5 on day 1.
   1. Transfer slices from the cryoprotection solution to 0.1 M PBS at room temperature. Rinse three times for 10 min each, with 0.1 M PBS.
   2. Incubate slices for 30 min in endogenous peroxidase blocking solution to inactivate endogenous peroxidase. Rinse 3 times, 10 min each, with 0.1 M PBS. Optionally, perform antigen retrieval (see section 5). Incubate slices for 20 min with 2 N HCl at 37 °C. Rinse in 0.1 M borate buffer (8.5 pH) for 10 min. Rinse 3 times for 10 min each, with ice-cold 0.1 M PBS.
   3. Incubate slices for 2 h at room temperature with PBS++ (blocking solution). Incubate with anti-BrdU primary antibody (mouse host) at concentration of 1:250 in PBS+ overnight at 4 °C.
   4. On day 2, rinse the slices 3 times for 10 min each with 0.1 M PBS.
   5. Incubate with 1:250 HRP-conjugated secondary antibody (anti-mouse) in PBS+ for 2‒4 h at room temperature. Rinse 3 times for 10 min each with 0.1 M PBS.
   6. Transfer slices to DAB Peroxidase (HRP) Substrate solution and incubate for 2‒10 min. When slices become dark grey, visualize the tissue with a magnifying glass or a microscope. If positive cells are present, rinse 3 times (for 15 min each) with tap water (to reduce background). Wash 3 times for 10 min each with 0.1 M PBS.
   7. Carefully mount slices on gelatinized slides using a soft brush, air dry overnight at room temperature. Counterstain (see section 7.1), add permanent mounting medium and place coverslips. Store at 4 °C for up to 6 months.
2. Detection of BrdU using peroxidase reaction with the avidin-biotin-peroxidase complex
   NOTE: Perform steps 4.2.1 to 4.2.5 on day 1.
   1. Transfer slices from the cryoprotection solution to 0.1 M PBS to bring to room temperature. Rinse 3 times for 10 min each with 0.1 M PBS.
   2. Incubate for 30 min with endogenous peroxidase blocking solution to inactivate endogenous peroxidase. Rinse 3 times for 10 min each in 0.1 M PBS. Optionally, perform antigen retrieval (see section 5).
   3. Incubate for 20 min with 2 N HCl at 37 °C. Rinse in 0.1 M borate buffer (pH 8.5) for 10 min. Wash 3 times for 10 min each with ice-cold 0.1 M PBS.
   4. Incubate for 2 h at room temperature in PBS++ (blocking solution).
   5. Incubate with anti-BrdU primary antibody (mouse host) 1:250 in PBS+ overnight at 4°C.
   6. On day 2, rinse 3 times for 10 min each with 0.1 M PBS.
   7. Incubate with 1:250 biotinylated secondary antibody (anti-mouse) in PBS+ for 2‒4 h at room temperature. Rinse 3 times for 10 min each with 0.1 M PBS.
   8. Incubate in the ABC solution for 1 h at room temperature. Rinse 3 times for 10 min each with 0.1 M PBS.
   9. Transfer slices to DAB peroxidase (HRP) substrate solution and incubate for 2‒10 min. When slices become dark grey, visualize the tissue with a magnifying glass or a microscope. If positive cells are present, rinse 3 times (15 min each) with tap water (to reduce background) followed by 3 times with 0.1 M PBS wash for 10 min each.
      NOTE: Solution must be prepared immediately before use. Care should be taken to avoid brain slices sticking to each other due to irregular dark spots in the tissue.
   10. Carefully mount slices on gelatinized slides using a soft brush and then air dry overnight at room temperature.
   11. Counterstain if needed (see step 7.1), add permanent mounting medium and place coverslips. Store at 4 °C for up to 6 months.
3. Detection of BrdU by immunofluorescence using labeled Streptavidin-Biotin (LSAB) amplification
   NOTE: Perform steps 4.3.1 to 4.3.4 on day 1.
   1. Transfer slices from the cryoprotection solution to 0.1 M PBS at room temperature. Rinse 3 times for 10 min each with 0.1 M PBS. Optionally, perform antigen retrieval (see section 5).
   2. Incubate for 20 min in 2 N HCl at 37 °C. Rinse in 0.1 M borate buffer (8.5 pH) for 10 min. Rinse 3 times for 10 min each in ice-cold 0.1 M PBS.
   3. Incubate for 2 h at room temperature in PBS++ (blocking solution). Incubate with 1:250 anti-BrdU primary antibody (mouse host) in PBS+ overnight at 4 °C.
   4. On day 2, rinse 3 times for 10 min each with 0.1 M PBS.
   5. Incubate with 1:250 biotinylated secondary antibody (anti-mouse) in PBS+ for 2‒4 h at room temperature. Rinse 3 times for 10 min each with 0.1 M PBS. Incubate with 1:250 fluorochrome-conjugated streptavidin (Cy3) in PBS (do not use serum) for 1‒2 h at room temperature. Rinse 3 times for 10 min each with 0.1 M PBS.
      NOTE: Serum can contain biotin and should not be added to diluents. Instead, use PBS containing 0.3% of Triton X-100.
   6. Carefully mount slices on gelatinized slides using a soft brush, air dry overnight at room temperature, or mount immediately with an appropriate mounting medium.Counterstain (see step 7.2), add permanent mounting medium and place coverslips. Store at 4 °C for up to 6 months.
4. Detection of BrdU by indirect immunofluorescence
   NOTE: Perform step 4.4.1 to 4.4.4 on day 1.
   1. Transfer slices from the cryoprotection solution to 0.1 M PBS until it reaches room temperature. Rinse 3 times for 10 min each with 0.1 M PBS. Perform antigen retrieval if required (optional, see section 5).
   2. Incubate for 20 min in 2 N HCl at 37 °C. Rinse in 0.1 M borate buffer (8.5 pH) for 10 min. Rinse 3 times for 10 min each with ice-cold 0.1 M PBS. Incubate for 2 h at room temperature with PBS++ (blocking solution). Incubate with 1:250 anti-BrdU primary antibody (mouse host) in PBS+ overnight at 4 °C.
   3. On day 2, rinse 3 times for 10 min each with 0.1 M PBS.
   4. Incubate with 1:250 fluorochrome-conjugated secondary antibody (anti-mouse) in PBS+ for 2‒4 h at room temperature. Rinse 3 times for 10 min each with 0.1 M PBS.
   5. Carefully mount slices on gelatinized slides using a soft brush, air dry overnight at room temperature, or mount immediately with an appropriate mounting medium. Counterstain (see step 7.2), add permanent mounting medium and place coverslips. Store at 4 °C for up to 6 months.

5. Antigen retrieval (optional)

NOTE: Antigen Retrieval is an optional step intended to correct the loss of antigenicity caused by fixation that modifies the tertiary and quaternary structure of many antigens, making them undetectable by antibodies. This step can be added to the original protocol.

1. In a microwave or water bath, pre-heat 10 mM sodium citrate buffer (SCB) pH 6 solution to 90‒95 °C (depending on altitude, the solution starts boiling around this temperature). Fill 80% of a 50 mL conical tube (40 mL) with pre-heated SCB. Transfer slices to mesh inserts into the conical tube with SCB. Cover the tube with a screw cap with holes made with an 18‒20 G needle.
2. Keep slices for 30 min in SCB at 80‒85 °C alternating warming cycles in the microwave at the minimum power level. If needed, refill the conical tube with SCB. Transfer slices immediately after along with the mesh inserts into ice-cold 0.1 M PBS and rinse 3 times for 10 min each.

6. Multiple immunostainings (optional)

NOTE: See the introduction section for the rationale behind this step.

1. Simultaneous multiple immunostainings
   1. Prepare a cocktail with the primary antibodies to the target (e.g., mouse anti-BrdU, and rabbit anti -GFAP) in PBS+. Use different hosts for each primary antibody used. Incubate overnight at 4 °C. Continue with the same next steps for each protocol.
   2. Prepare a cocktail with the corresponding secondary antibodies for each primary antibody used (e.g., goat anti-mouse FITC, goat anti-rabbit TRITC) in the same diluent solution for each protocol. Continue with the same next steps for each protocol. Ideally, use secondary antibodies that come from the same hosts to avoid cross-reaction.
2. Sequential multiple immunostainings.
   1. Follow the protocol for the first antibody target (e.g., mouse anti-BrdU) and stop before the mounting the slices. Incubate for 2 h at room temperature with PBS++ (blocking solution).
   2. Incubate the second primary antibody (e.g., rabbit anti -GFAP) in PBS+ overnight at 4 °C. Follow the next steps for each protocol, including the incubation of the second secondary antibody (e.g., goat anti-rabbit TRITC). Continue with the next steps for each protocol to the end.

7. Counterstaining (optional)

1. For protocols using peroxidase reaction, pre-heat the cresyl violet solution to 60 °C. Hydrate the slides with ddH₂O for 1 min. Incubate the slides in the hot cresyl violet solution for 5‒20 min.
   1. Rinse the slides with ddH₂O for 1 min. Rinse the slides with 70%, 80%, 90% and 100% ethyl alcohol for 1‒3 min each. Rinse the slides with xylene for 1‒3 min.
   2. Add permanent hydrophobic mounting medium and place coverslips.
      NOTE: Store at 4 °C for up to 6 months. Self-made mounting medium containing PVA (Polyvinyl alcohol)-DABCO can be employed.
2. For protocols using immunofluorescence, add a small volume (25‒50 μL) of hydrophilic mounting medium with DAPI, propidium iodide or similar. Seal around the perimeter with nail polish or a plastic sealant. Store at 4 °C for up to 6 months.

8. Imaging and analysis

NOTE: See Table 2 for microscope setup specifications. Usually, counting the stained new cells is done using the peroxidase reaction stained slices (cheaper method), but it can also be performed using immunofluorescence.

1. To quantify cells, first, identify the dentate gyrus properly with the 4x magnification lens (for further instructions on DG anatomical detail, see Amaral et al.¹⁶).
   1. Search the granular cell layer of the dentate gyrus for nuclei labeled with BrdU (using the 40x magnification lens). Perform cell search exhaustively along the z-axis since new cells can be distributed in different layers (see Video 1).
   2. Select an interval section for cell searching all over the dentate gyrus (e.g., every 6th section of tissue, equivalent to every 240 μm).
   3. Count all BrdU positive cells. The morphology of the labeled nucleus can change depending on how much BrdU the cell incorporated (see Figure 3 as a guide). Move slowly over the z-axis to quantify all several nuclei that integrate a cluster (see Video 2).
   4. Multiply the total number of counted cells with the interval section selected (e.g., 6) to estimate the total number of BrdU-labeled new cells.
   5. Ideally, in a regular experiment, count at least ten sections per animal and at least five animals per group.
2. Image deconvolution (optional)
   NOTE: Refer to the introduction section for important information about this step. This procedure needs monochromatic images (grayscale). Transform color images to grayscale. If the images are an RGB composite, first split the channels and merge them as a single image (not composite), then transform to 8-bit grayscale.
   1. Create a z-stack file from micrographs.
   2. Create a point spread function (PSF) file opening the Diffraction PSF 3D plugin (https://imagej.net/Diffraction_PSF_3D) from the option Plugins menu. Fill out all the required data (see Table 2). Press OK and save the file.
   3. Open the DeconvolutionLab2⁹ plugin from the option Plugins menu (http://bigwww.epfl.ch/deconvolution/deconvolutionlab2/). Drag the matched z-stack image and PDF file to the corresponding window slot.
   4. Select the deconvolution algorithm (e.g., Richardson-Lucy) and the number of iterations (e.g., 20). Press RUN.
   5. Combine the deconvoluted images into a single z-stack image selecting Stacks from the Image menu at the top. Then click Z Project. Select Max Intensity from the Projection Type dropdown menu, press OK and save the file.
   6. Create an RGB image using the single z-stack image file created in the step above with the desired pseudocolor selecting Color from the Image menu at the top. Then click on Merge Channels. Set the corresponding image to the desire color channel from the dropdown menu. Uncheck the box Create Composite, press OK and save the file (see Figure 4).
   7. If there is more than one channel image, repeat the steps 8.2.1‒8.2.5. Create an RGB image file following the step 8.2.6, opening at least two image files and selecting different color channels for each image file (see Figure 4).