Axio imager m2 fluorescent microscope

We utilized the same flow cytometry antibodies for immunohistochemical staining of T cells, B cells, and macrophages. Three tissue sections around 2 mm rostral or caudal to the epicenter were selected. Sections were washed twice with TBST (50 mM Tris, 150 mM NaCl, 0.05% Tween 20, PH 7.6) for 15 min at room temperature. Sections were blocked using 5% normal mouse serum in TBST for 1 h at room temperature and incubated with antibodies against cell surface markers for B cells (CD45RA), T cells (CD3), and macrophages (Iba-1, CD68, CD86, CD163),1:500 for Iba-1 and 1:100 for the rest of the antibodies overnight at 4 °C. Sections were then washed twice with TBST and incubated with their corresponding intracellular (IL-10 for B cells, IL-10, and FoxP3 for T cells) antibodies. FoxP3 antibody was diluted in an antibody solution containing 1% BSA, 0.1% cold water fish skin gelatin, 0.5% Triton X-100, and 0.05% sodium azide in TBS. Images were taken at × 20 and × 63 using Axio Imager M2 fluorescent microscope (Zeiss) and were processed using ZEN software (Zeiss).

Animals were injected with anesthesia cocktail (ketamine/xylazine/acepromazine, 50/5/0.5 mg/kg), and under deep anesthesia animals were perfusion fixed with 4% paraformaldehyde. After cryoprotection in 800 mosmol/L sucrose and freezing in Optimal Temperature Cutting (OCT) compound, 5‐μm sections were cut. Sections were incubated overnight at 4°C with anti‐anti‐TRPV5 (1:100, Alomone) or anti‐calbindin (1:500, Swant). Sections were incubated in secondary antibody at 1:2000 for 1 h at room temperature [Alexa Fluor 488 donkey anti‐rabbit (Life Technologies A21206) or Alexa Fluor 555 donkey anti‐mouse (Life Technologies A31570)]. All sections were mounted with ProLong Diamond Antifade Mountant (ThermoFisher Scientific P36970). Images were captured with a ZEISS AXIO Imager M2 fluorescent microscope.

For quantification of cell density and neurofilament staining, fluorescence microscopy and image capture were performed using a Zeiss Axio Imager M2 fluorescent microscope with standard epifluorescence optics and Zen Blue software (Carl Zeiss AG, Germany). To avoid bias, fields of view (FoV) were selected in the blue (DAPI) channel and images were captured (10 images from across each coverslip) at x20 magnification in the red, far red, green and blue channels. Representative images for illustration were obtained using the same microscope and software or with a Zeiss LSM 880 Confocal Microscope using a Zeiss Plan-Apochromat 63 × /1.4 oil immersion objective and Zen Black software.

All SNpc containing sections from the 3-month-old and the PBS or PFF inoculated mice were analysed stereologically using an Axio Imager M2 fluorescent microscope (Zeiss) and Stereo Investigator 10.0 software (MBF Bioscience). The SNpc region of interest was outlined for sampling, and live focus and an optical fractionator stereological probe (box size = 30 × 30 × 30 μm³ sampling 2 cells on average per box; grid sampling array 137 μm × 101 μm with 12.8 ± 0.5 boxes sampled) used to obtain an unbiased stereological sample within this region. Only those neurons whose nuclei fell entirely inside the probes frame or touching the green inclusion borders of the probe were counted. Repeated measures in 25% of sections by the same assessor varied by <6%. For the analysis of brain tissue from 18-month-old mice, a stereological fractionator technique was used with images of the entire SNpc in spaced sections captured at 10× magnification and the number of all TH immunofluorescent neurons determined within each section using the manual count tool in Adobe Photoshop. For each aged mouse, the total number of TH immunoreactive neurons was estimated by multiplying the sum of the number counted per section ×6. Repeated measures in 25% of sections by the same assessor varied by <8%. For all studies, TH neuron counts in KI mice were expressed as a percent of wild type mice, which was set at 100%.

Immunofluorescence of viral capsid protein VP2 was determined on formalin-fixed, paraffin-embedded sections using viral capsid VP2 antibody (QED Bioscience Inc) which binds to capsid protein VP2 of HRV16, HRV1A and HRV39. RV-VP2 antibody was raised in mouse and used in 1:5,000 dilution and labeled with goat anti-mouse IgG antibody conjugated to Alexa Fluor 488 (Cell signaling Technology; #4408) in 1:1,000 dilution. Following staining, a minimum of 5 images were captured using Axio Imager M2 fluorescent microscope (Carl Zeiss AG, Oberkochen, Germany) and a representative image from each group was reported.