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3 protocols using hrp conjugated anti rabbit igg antibody

1

UBAP2L Localization in ALS Spinal Cord

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Human spinal cord paraffin sections from clinically and histopathologically characterized ALS patients and neurologically healthy individuals were obtained from the Sheffield Brain Tissue Bank. Consent was obtained from all subjects for autopsy, histopathological assessment, and research in accordance with local and national Ethics Committee–approved donation. Three control cases, three sALS, and three ALS-C9 cases were included in the study. Human spinal cord sections were 7 µm thick. Rehydrated sections were subjected to microwave antigen retrieval in sodium citrate buffer. After incubation with the primary anti-UBAP2L antibody overnight at 4°C, secondary HRP-conjugated anti-rabbit IgG antibody (Vector Laboratories) was applied for 1.5 h at RT. Vectastain Elite ABC Universal Plus Kit (Vector Laboratories) and DAB (Sigma-Aldrich) were used for detection. Images were taken with a 20× objective (UPLFLN 20×Ph/0.5) of a BX53 microscope equipped with DP73 camera and processed using cellSens Standard 1.9 software (all Olympus).
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2

Histological and Immunohistochemical Analysis of SARS-CoV-2 in Lung Tissue

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Histological and immunohistochemical assays were conducted as described51 (link),55 (link). Immediately after euthanasia, the left lung lobe was harvested and fixed by submersion in 10% phosphate-buffered formalin for overnight, followed by submersion in 70% ethanol for up to one week, which was enough to disinfect the viruses. Fixed tissue was manually embedded in paraffin, and sections were prepared at 4 µm thickness. Tissue sections were stained with hematoxylin and eosin (Richard Allan Scientific, San Diego, CA). For immunohistochemistry, antigen retrieval was performed with antigen unmasking solution (pH 6.0 citrate buffer, H-3300, Vector Laboratories, Newark, CA), followed by peroxide incubation. The tissue sections were first incubated with primary antibody, SARS-CoV-2 nucleocapsid protein rabbit mAb (26369, Cell Signaling, Danvers, MA) for overnight, and then with secondary antibody, HRP-conjugated anti-rabbit IgG antibody (MP7401, Vector Laboratories). The signal was then amplified using a DAB staining kit (NC9567138, Fisher Scientific, Waltham, MA) and it was counterstained with hematoxylin. Finally, tissues were tiled scanned using an Aperio AT2 slide scanner (Leica Biosystems, Deer Park, IL), and images were captured with an Aperio ImageScope (Leica Biosystems, Deer Park, IL).
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3

hnRNPA3 Immunohistochemistry in FUS-TG Mice

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Spinal cord sections (8 μm thick) from wild-type and 4-month old symptomatic FUS-TG mice (Shelkovnikova et al., 2013a (link)) were used. After rehydration, sections were subjected to microwave antigen retrieval in sodium citrate buffer (pH 6.0) and blocked using 10% goat serum in PBS/T. Sections were incubated with the primary anti-hnRNPA3 antibody (Proteintech, 25142–1-AP) overnight at 4 °C and secondary HRP-conjugated anti-rabbit IgG antibody (Vector Laboratories) for 1.5 h at RT. Signal was detected using Vectastain® Elite ABC Universal Plus Kit (Vector Laboratories) and 3,3′-diaminobenzidine (DAB, Sigma). Images were taken using BX57 microscope (Olympus) and ORCA-Flash 4.0 camera (Hamamatsu).
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