Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

All experimental procedures were performed with the approval of the animal experiment ethics committee at the University of Tokyo and according to the University of Tokyo guidelines for the care and use of laboratory animals.

1. Preparation of Solutions

1. Prepare 0.01 M Phosphate-Buffered Saline (PBS): add a PBS tablet (0.14 M NaCl, 0.0027 M KCl, 0.010 M PO₄^3-, pH 7.4) to 1 L distilled water and stir at RT until it is completely dissolved.
2. Prepare 4% paraformaldehyde (PFA) in PBS: add 4 g of PFA to 100 mL PBS. Stir the solution at 60 °C until it is completely dissolved.
   1. After dissolving the PFA in PBS, adjust the pH of the solution to 7.4 with 1 N sodium hydroxide (NaOH). Then, cool on ice and filter the solution through a filter paper to remove any particulate matter. Store at 4 °C.
      Caution: Take appropriate care when using these reagents. Handle with care and use gloves, safety goggles, and lab coat under a chemical hood.
      NOTE: Use fresh 4% PFA solution prepared within 2 d.
3. Prepare 0.01 M PBS supplemented with 0.3% Triton X-100 (PBST): add 3 mL of Triton X- 100 to 997 mL of PBS. Stir the solution at RT until it is completely dissolved.
4. Prepare 1% and 5% blocking solution: add 10 g of skim milk to 200 mL PBST to prepare 5% blocking solution. Stir at RT until it is completely dissolved. Dilute 5% blocking solution five times with PBST to prepare 1% blocking solution.

2. Preparation of Parasagittal OB Sections

1. Use animals between 1-2 weeks of age. These age groups are suitable for the following experiments because glomeruli are clearly visible and the brain can be cut with the skull.
2. Anesthetize the animal with an intraperitoneal injection of pentobarbital (50 mg/kg body weight). Perfuse the animal transcardially with ice-cold 4% PFA in PBS. Handle with care and use gloves, safety goggles, and lab coat under a chemical hood.
3. After perfusion, cut off the head with scissors and remove the skin carefully. Then, insert a blade of the scissors into the space between the upper and lower teeth and cut horizontally to remove the lower jawbone. Trim away excess tissue with forceps and scissors to leave the olfactory tissue including the OB and OE.
   NOTE: Do not remove the skull surrounding the OB as this retains the medial glomeruli aligned vertically straight.
4. Immerse the olfactory tissue in PBS to remove the air in the nasal cavity. Cut off the posterior part of the brain vertically and place the olfactory tissue on the bottom of the embedding mold with the cutting face down. Fill the mold with Optimal Cutting Temperature (O.C.T.) compound and then immerse the mold into liquid nitrogen.
   1. After the tissue in the compound is completely frozen, incubate it in the cryostat at -20 °C for 1 h.
5. Make serial parasagittal sections (10 µm) of the OB with a cryostat and collect them by sticking to MAS-coated glass slides. After sticking, dry the slides immediately with a blow dryer.

3. Day 1: Quadruple Immunostaining of the OB Slices 

1. Wash the slides for 5 min with PBS at RT. Repeat 3 times.
2. Block the nonspecific binding sites by incubating the slides for 1 h with the 5% blocking solution at RT.
3. Incubate the slides O/N with a cocktail of primary antibodies (400 µL each slide) in the 1% blocking solution at RT. Use the following primary antibodies: guinea pig anti-Kirrel2 antibody (1:1000), goat anti-Semaphorin-7A (Sema7A) antibody (1:500), rat anti-OL-protocadherin (OLPC) antibody (1:500), and mouse anti-vesicular glutamate transporter2 (VGLUT2) antibody (1:500).

4. Day 2: Quadruple Immunostaining of the OB Slices 

1. Discard the primary antibody solution and wash the slides for 5 min with PBST at RT. Repeat 3 times.
2. Incubate the slides for 1 h with a cocktail of secondary antibodies (400 µL each slide) in PBS at RT. Use the following secondary antibodies: donkey anti-mouse Alexa Fluor 405 (1:400), donkey anti-goat Alexa Fluor 488 (1:400), donkey anti-guinea pig Alexa Fluor 555 (1:400), and donkey anti-rat Alexa Fluor 647 (1:400).
   NOTE: All secondary antibodies should come from the same host species. Choose a different wavelength of fluorescence of secondary antibodies for each primary antibody to ensure no overlap of wavelengths.
3. Discard the solution and wash the slides for 5 min with PBS at RT. Repeat 3 times.
4. Mount coverslips on the slides with the mounting medium. For this, apply 2 drops of the mounting medium on each slide, and then, put a coverslip by removing the air bubbles.

5. Intensity Measurements

1. Obtain fluorescent images with a fluorescence microscope. Use the following filter cubes: DAPI filter cube (360/40 nm excitation, 460/50 nm emission, and 400 nm dichroic mirror) for Alexa Fluor 405 signals, GFP filter cube (470/40 nm excitation, 525/50 nm emission, and 495 nm dichroic mirror) for Alexa Fluor 488, TRITC filter cube (545/25 nm excitation, 605/70 nm emission, and 565 nm dichroic mirror) for Alexa Fluor 555, and Cy5 filter cube (620/60 nm excitation, 700/75 nm emission, and 600 nm dichroic mirror) for Alexa Fluor 647.
   Note: Adjust the exposure time to obtain fluorescent signals of the image without saturation.
2. Define the glomerular structure by immunofluorescence signals of VGLUT2. Measure staining intensities of axon-sorting molecules within glomeruli with ImageJ.

6. Data Analysis

1. Subtract background signals from the staining intensities of axon-sorting molecules.
2. Prepare the expression data matrix, which has columns composed of axon-sorting molecules and rows composed of the glomeruli.
3. Perform a principal component analysis (PCA) using the prepared expression dataset. Then, obtain the PCA scores, contribution ratios, and factor loadings of each principal component.