Goat anti rat igg

For all experiments except blood vessel tracing, animals were anesthetized with diethyl ether until complete areflexia and transcardially perfused with heparinized Krebs solution until complete blood clearance, followed by 4% PFA in PBS. For blood vessel tracing, animals were anesthetized and decapitated without transcardiac perfusion; the brain was rapidly dissected out (3–6 min from decapitation to fixation) and immersed in 4% PFA. All samples were postfixed overnight in 4% PFA and then cryoprotected in 30% sucrose solution until sinking.
Cryoprotected samples containing the intact temporal bone were decalcified with buffered 10% EDTA in PBS (daily changed) until softening of the squamous temporal bone (3–4 weeks). Decalcified samples were embedded in 5% gelatin, trimmed, and halved. After trimming, gelatin was mechanically removed from the sample and clearing was performed using the iDISCO+ protocol (Renier et al., 2016 (link)). Samples (shown in Supplementary Figure S1 before and after clearing) were immunolabeled using rabbit anti-Iba1 (WAKO, 1:200) for macrophages/microglia, sheep anti-transthyretin (TTR; Abcam, 1:250) for CP epithelium and goat anti-rat IgG (Thermo Fisher, 1:200) for blood vessel lumen (as in, Liebmann et al., 2016 (link)). Species-matched Alexa-conjugated donkey secondary antibodies (Life Technology) were used at 1:200.

Immunolocalization of OSX, RANKL and ALP was performed using rabbit anti-mouse antibodies (anti-OSX #ab22552, anti-RANKL #ab37415, Abcam, Cambridge, UK; anti-ALP # 11187–1-AP, Proteintech, Rosemont, Illinois, USA) applied at a dilution of 1:200 in PBS + 1% turn BSA, overnight at 4 °C. After repeated washing with PBS, sections were incubated for 30 min with biotin-conjugated swine anti-rabbit IgG (#P0217, Agilent, Santa Clara, CA, USA) 1:200 in PBS + 1% BSA and then exposed for 30 min to peroxidase-conjugated ExtrAvidin (#P0217, Agilent) (1:50 in PBS + 1% BSA). The peroxidase reaction was developed using DAB substrate kit (SK-4100, Vector Laboratories, Burlingame, CA, USA).
Immunolocalization of LEPR, endomucin (EMCN) and alpha-smooth muscle actin (α-SMA) was performed using goat anti-LEPR (#AF497, R&D Systems, Minneapolis, MN, USA), rat anti-EMCN (#ab106100, Abcam) and rabbit anti-α-SMA (#ab5694, Abcam). Twenty-five μm-thick sections from gelatin-embedded samples were rehydrated with PBS and then immunostained overnight. After primary antibody incubation, sections were repeatedly washed with PBS and incubated with appropriate Alexa Fluor 647-conjugated (rabbit anti-goat IgG #A-21446, goat anti-rat IgG #A-21247, goat anti-rabbit IgG #A27040 Thermo Fisher Scientific) secondary antibodies for 1 h at room temperature. Nuclei were counterstained with TO-PRO3.

The antibodies used in this study are listed in Table 1. The source, catalogue number and dilution factor used for each antibody are provided. Additionally, we provide references for the antibody specificity tests and highlight which antibody was used in each figure. For non‐commercially sourced antibodies, antibodies were generated by the Division of Signal Transduction Therapy (DSTT), University of Dundee as described previously 20, 59. For HRP‐coupled secondary antibodies, goat anti‐rabbit‐IgG (cat.: 7074, 1:2,500) was from CST, and rabbit anti‐sheep‐IgG (cat.: 31480, 1:5,000), goat anti‐rat IgG (cat.: 62‐9520, 1:5,000) and goat anti‐mouse‐IgG (cat.: 31430, 1:5,000) were from Thermo Fisher Scientific.
For immunofluorescence studies, signal amplification was achieved using AlexaFluor‐594‐conjugated donkey anti‐sheep IgG (H+L) (cat.: A11058, Life Technologies, 1:300), AlexaFluor‐594‐conjugated goat anti‐rabbit IgG (H+L) (cat.: A11012, Life Technologies, 1:500) and goat anti‐mouse IgG (H+L) (cat.: A11005, Life Technologies, 1:500).

Tumor tissues were harvested and snap-frozen with optimum cutting temperature medium. The tumor tissue was then cut with cryotome, mounted on a slide, and stained using primary antibodies F4/80 (Abcam, cat. no. ab100790, 1:200 dilution) and CD8 (Abcam, cat. no. ab22378, 1:200 dilution) for 12 h at 4 °C. After adding fluorescently labeled secondary antibodies (goat anti-rabbit IgG (H + L; ThermoFisher, cat. no. A32733, 1:1000 dilution) and goat anti-rat IgG (H + L; ThermoFisher, cat. no. A18866, 1:600 dilution)), the slide was observed using CLSM (C2SI, Nikon, Japan).

HEK293T WT and DHX30-deficient cells were plated on glass coverslips coated with poly-L-Lysine. After 24 h, cells were heat stressed at 43.5 °C for 1 h, fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 (Sigma). Blocking was performed using 10% horse serum (HS). Rat monoclonal anti-ATXN2 was used as a primary antibody (1:10 in 2 % HS in PBS), followed by goat anti-rat IgG coupled to Alexa Fluor 647 (Thermo Fisher Scientific). Coverslips were mounted on glass microscope slides with ProLong Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific). Immunofluorescence images were acquired using a confocal microscope (Leica TCS SP5, 63x/1.25 objective) and processed with ImageJ software.