All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Perfusion and tissue processing
- Anesthetize mice using an intraperitoneal injection with ketamine (100 mg/kg) and xylazine (10 mg/kg). Pinch toes to confirm a lack of response before continuing.
- Transcardially perfuse with ~50 mL of ice-cold 0.1M Dulbecco's phosphate-buffered saline (DPBS) followed by 4% paraformaldehyde (PFA) in 0.1M phosphate buffer (PB).
CAUTION: PFA is toxic. Use personal protective equipment (PPE) when working with PFA.
- Remove the brains and immerse them in 4% PFA in 0.1 M PB at 4 °C for 24 h for post-fixation. Wash with DPBS 3x.
- Change to 15% sucrose solution in 0.1 DPBS overnight and then 30% sucrose solution in 0.1 M DPBS for another 48 h at 4 °C.
- Remove the tissue from the sucrose solution and freeze in a cryotome chamber preset to a temperature of -20 °C. Frozen samples can be stored in sealed tubes at -80 °C until sectioning.
- Embed tissues in optimum cutting temperature embedding medium (see Table of Materials) and mounted on a specimen disc. Cut serial 30 µm thick sections in a coronal plane using a cryotome and collect all the sections consequently in 24 well culture plates filled with cryoprotectant solution (30% glycerol, 30% ethylene glycol, 40% DPBS; Figure 1A–C). Store in a -20 °C freezer.
NOTE: Thicker sections (e.g., 50 µm) are preferable when feasible. Make sure that the sections are kept in cutting order and that all sections are completely in cryoprotectant. A 96-well plate can be used as an alternative to 24 well plates to collect sections.
- Cover the wells with plate sealer to prevent evaporation and drying. Store plates at -20 °C until further use. Sections stored at -20 °C are stable for several months for choline acetyltransferase (ChAT) immunohistochemistry.
2. Systematic section selection for IHC
NOTE: A pilot study should be done to know the total number of sections required to achieve an acceptable coefficient of error (CE). The CE value is an expression of the total amount of error in the sampling procedure. The lowest value represents the minimal error, and a CE value lower than 0.1 is considered acceptable by the software used here (see Table of Materials).
- Identify the first and last sections for each brain by comparing morphological features with a standard mouse brain atlas such as Franklin and Paxinos. Nucleus basalis of Meynert or NBM begins at bregma -0.0mm and ends at -1.6 mm. Therefore, about 50 sections contain NBM. The total number of sections is one of the parameters required during stereology. The selection of every 8th section (SSF = 1/8) gave a total of 6–7 sections for the analysis and yielded an acceptable CE for both volume estimation and fiber length estimation in our pilot study.
- Randomly start with one of the first eight sections and then systemically sample every 8th section until the last posterior section containing NBM (Figure 1C).
3. Immunohistochemistry
- Transfer the cryopreserved sections to room temperature in cryoprotectant and then 0.1M phosphate buffer (PB) in 6 well plates.
- Wash 3x in PB.
- Incubate in 0.3% H2O2 in methanol for 15 min.
- Wash 3x in Tris-buffered saline (TBS).
- Incubate in 0.25% Triton X-100 in TBS for 30 min.
- Block with 10% normal bovine serum (NBS) in 0.1% Triton X-100 in TBS for 30 min.
- Incubate with a 1:1,000 dilution of goat anti-human ChAT primary antibodies (see Table of Materials) in TBS with 0.1% Triton X-100 and 1% NBS for 48 h at 4 °C.
- Wash 3x in TBS at room temperature. Perform all further incubation and washing at room temperature.
- Incubate with biotinylated bovine anti-goat secondary antibody (see Table of Materials) for 1 h.
- Wash 3x in TBS.
- Incubate with the avidin-biotin–peroxidase complex (see Table of Materials).
- Wash 3x in TBS.
- Develop using an enhanced DAB peroxidase substrate solution (see Table of Materials) according to the manufacturer's recommendations.
CAUTION: DAB is a suspected carcinogen. It is toxic by contact and inhalation. Use PPE when working with DAB.
- Wash the section a couple of times with distilled water and keep in Tris pH = 7.6.
- Mount the sections on the gelatinized slides. All sections from one tissue can be put on the same slide. Air dry the sections, dehydrate 2x for 5 min each with 70%, 90%, 95%, and 100% ethanol, and then clear the sections with two 10 min xylene washes. Coverslip the sections using a mounting medium (see Table of Materials).
NOTE: The processing time of dehydration and clearing affects the thickness of the sections. Therefore, the same conditions should be used for all sections. In the current study, the mean value for the final thickness was 21.11 ± 0.45 µm.
- Keep the sections in the fume hood to dry. The dry sections are ready for stereology.
