Dp71 digital camera

Mouse brains and spinal cords were collected following intra-cardiac perfusion with PBS/heparin. Tissues were fixed with 70% ethanol/150 mM NaCl for 48 h followed by paraffin processing. Paraffin-embedded tissue sections were deparaffinized and hydrated through a series of graded ethanol solutions followed by washing with 0.1 M Tris, pH 7.6. The sections were blocked with 2% FBS in 0.1 M Tris, pH 7.6. Immunohistochemical detection was done using avidin-biotin complex (ABC) system (Vectastain ABC Elite Kit, Vector Laboratories, Burlingame, CA) and immunocomplexes were visualized with the chromogen 3,3′-diaminobenzidine (DAB). Sections were counterstained with hematoxylin. Slides were scanned using an Aperio ScanScope CS (Aperio Technologies Inc., Vista, CA) and images acquired using the ImageScopeTM software (Aperio Technologies Inc.). Quantification of immunostaining was done using the Pixel count Program (ImageScope, Aperio Technologies). For immunofluorescence detection, sections were incubated with secondary antibodies conjugated to Alexa fluor 594 or Alexa fluor 488 (Invitrogen, Eugene, OR) followed by Sudan Black treatment and staining with DAPI (Invitrogen, Eugene, OR). The sections were coverslipped with Fluoromount-G (Southern Biotech, Birmingham, AL) and visualized using an Olympus BX51 microscope mounted with a DP71 Olympus digital camera.

Paraffin-embedded slides were deparaffinized, rehydrated, and incubated at room temperature in 0.06% KMnO₄ solution for 10 min. Slides were incubated in 0.0001% Fluoro-Jade C (EMD Millipore) dissolved in 0.1% acetic acid for 10 min with gentle shaking. Slides were washed, counterstained with DAPI, and dried on a slide warmer at 50 °C for 5 min. Slides were cleared in xylene for 1 min and then mounted using Cytoseal 60 mounting medium (Fisher Scientific). Slides were imaged using an Olympus BX51 microscope mounted with a DP71 Olympus digital camera. One slide was stained per mouse (each slide contained 2–4 lumbar spinal cord coronal sections). The total number of Fluoro-Jade C-labeled puncta was manually counted for all the spinal cord sections of any one sample and then averaged for each cohort.

After euthanasia and perfusion, the brain and spinal cord from perfused animals were fixed in 30% sucrose solution containing 4% paraformaldehyde. Tissues were embedded in paraffin blocks, cut into 4 µm using a Rotary Microtome (Leica RM2235, Leica, Nussloch, Germany), and mounted on glass slides. Sections of spinal cord and brain tissues were then deparaffinized and rehydrated in a graded ethanol series. Immunohistochemistry was performed as previously described 19 (link). Briefly, tissues were incubated in 3% hydrogen peroxide solution and then subjected to antigen retrieval at 100 °C for 10 minutes in citrate buffer. Tissues were blocked in normal goat serum for 20 minutes at room temperature and then incubated with anti-pTDP-43 (phosphorylated at Ser409/Ser410) antibody (mouse, Millipore, 1:800) at 4 °C for 24 hours. Then, the labeling was detected using a Streptavidin-Peroxidase kit (Bioss, China) and visualized with diaminobenzidine. Images were captured using an Olympus IX51 microscope mounted with a DP71 Olympus digital camera.

The mice were transcardially perfused with a saline solution containing 0.5% sodium nitrate and heparin (10 U/ml). The lungs and colons were removed from the perfused mice, fixed with 10% neutral-buffered formalin (IMEB Inc., San Marcos, CA, USA), and embedded in paraffin. A section of the lung (4-µm thick) and transverse colons (7-µm thick) from each mouse were stained with hematoxylin and eosin and visualized on an Olympus BX51 microscope (Olympus, Tokyo, Japan) equipped with a DP71 digital camera (Olympus). A total histological score of the lung was calculated for each mouse as described previously (18 (link)). Briefly, the lung sections were scored from 0 to 5 by readers according to the following criteria: 0 = normal; 1 = very mild; 2 = mild; 3 = moderate; 4 = marked; 5 = severe inflammation. The lung sections were examined under 200× magnifications to quantify the mean alveolar airspace (MAA). MAA was a quantitative assessment of lung structure that was determined by dividing the sum of the alveolar airspace areas divided by the number of identified alveoli using Image Pro-Plus 5.1 software (Media Cybernetics, Inc., Silver Spring, MD, USA). The colon sections were assigned a score from 0 to 3 as described previously (23 (link)). The histological analyses were performed in a blinded fashion.