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6 protocols using vectashield hardmount

1

Spinal Cord Immunohistochemistry Staining Protocol

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Mice were deeply anaesthetized by i.p. injection of pentobarbital and perfused transcardially with PBS, followed by ice-cold 4% paraformaldehyde/PBS. The L3–4 (transverse) segments of the spinal cord were removed, postfixed in the same fixative for 3 h at 4 °C, and placed in 30% sucrose solution for 24 h at 4 °C. Transverse L4 spinal cord sections (30 μm) were incubated in blocking solution (3% normal goat serum or normal donkey serum) for 2 h at room temperature and then incubated for 48 h at 4 °C with primary antibodies: polyclonal rabbit anti-PAX2 (1:1000, Invitrogen), monoclonal rat anti-GFAP (1:2000, Invitrogen), monoclonal mouse anti-NeuN (1:2000, Millipore), polyclonal rabbit anti-Iba1 (1:5000, Wako), and polyclonal rabbit anti-c-Fos (1:5000, Santa Cruz). Following incubation, tissue sections were washed and incubated for 3 h at room temperature in secondary antibody solution (Alexa Fluor 488 and/or 405, 1:1000, Molecular Probes). The tissue sections were washed, slide-mounted and subsequently coverslipped with Vectashield hardmount (Vector Laboratories). Three to five sections from the L4 spinal cord segments of each mouse were randomly selected and analysed using a LSM 700 Imaging System (Carl Zeiss).
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2

Confocal Imaging and Tracing of Interneurons

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Slices were fixed in 4% paraformaldehyde (Boston BioProducts) and incubated with streptavidin Alexa Fluor 633 (Life Technologies, Invitrogen) in phosphate buffered saline (PBS) with Triton-X 100 as previously described (Bell et al., 2011 (link)). Processed slices were then reconstructed using a Zeiss LSM 710 confocal microscope (Carl Zeiss, Jena, Germany). Alexa Fluor 633 was excited with the 633 nm line of a HeNe 5 mW laser and cells were visualized using a 20× dry lens (0.8 N.A., voxel dimensions 0.2 × 0.2 × 1.1 µm). The imaged interneurons were traced using the Autoneuron module within the Neurolucida program (MBP, Burlington, VT). For amplification of YFP-labeled interneurons, 1:200 dilution of rabbit anti-GFP conjugated to Alexa Fluor 488 (Life Technologies, Invitrogen) in goat blocking buffer (10% normal serum, 2% bovine serum albumin, 0.4% Triton-X 100 in 0.1 M phosphate buffer) was added to fixed and washed slices for overnight incubation. Before and after primary and secondary antibody incubations, slices were washed in PBS. Slices were mounted in either Prolong Gold® (Life Technologies, Invitrogen) or VECTASHIELD® hard mount (Vector Laboratories).
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3

Immunohistochemical Analysis of Spinal Cord and DRG

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Mice were deeply anaesthetised by i.p. injection of pentobarbital and perfused transcardially with phosphate buffered saline (PBS), followed by ice-cold 4% (w/v) paraformaldehyde/PBS. The L4 segments of the spinal cord, or the L4 DRG were removed, postfixed in the same fixative for 3 h at 4 °C, and placed in 30% (w/v) sucrose solution for 24 h at 4 °C. Transverse L4 spinal cord sections (30 μm) and L4 DRG sections (15 μm) were incubated in blocking solution [3% (v/v) normal goat serum] for 2 h at room temperature and then incubated for 48 h at 4 °C with primary antibodies: rabbit polyclonal anti-Iba1 (1:5000, Wako), and rat monoclonal anti-CD11b (1:1000, Serotec), rat monoclonal anti-CD31 (1:200, BD Pharmingen) and hamster monoclonal anti-CD3 (1:100, eBioscience). Following incubation, tissue sections were washed and incubated for 3 h at room temperature in secondary antibody solution (Alexa Fluor 546 and Alexa Fluor 405, 1:1000, Molecular Probes, OR, USA). The tissue sections were washed, slide-mounted and subsequently coverslipped with Vectashield hardmount (Vector Laboratories). Three to five sections from the L4 spinal cord and DRG of each mouse were randomly selected and analysed using an LSM700 Imaging System (Carl Zeiss).
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4

CTLA-4 Expression in Lung Cancer Xenografts

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Fluorescence immunohistochemistry was performed to confirm CTLA-4 expression levels in ex vivo lung cancer xenograft tissues. Tumor tissues were collected, frozen in Optimal Cutting Temperature compound (Sakura Finetek), and sectioned in 5 μm slices. For staining, slides were dried for 15 min at room temperature and fixed in 4% paraformaldehyde (Santa Cruz Biotechnologies), then washed twice with 1X PBS. Slides were then permeabilized in 0.2% Triton X (Sigma-Aldrich) in PBS for 15 min, washed with PBS, and blocked for 2 h in blocking solution at room temperature (5% donkey serum, 0.3% Triton X in PBS). Primary incubation with ipilimumab (25 μg/mL) in the blocking solution was performed overnight at 4°C. To reduce background, slides were then washed twice with 0.2% Triton X for 30 min and washed with PBS prior to incubation with goat anti-human 800CW secondary dye (LI-COR) for 1 h. Slides were subsequently washed twice with 0.2% Triton X for 20 min and twice with PBS for 5 min, mounted with VectaShield hard mount containing DAPI (Vector Laboratories) and coverslipped. Imaging was performed with a Nikon A1R confocal microscope.
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5

Multiplex Immunofluorescence Staining and Analysis

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Multiplexed immunofluorescent staining and multispectral image analysis were performed as previously described [42 (link)]. In brief, 4‐mm‐thick formalin‐fixed paraffin‐embedded slides were deparaffinized in a Leica auto‐stainer (Leica Biosystems, Nussloch, Germany), and citrate buffer pH 6.0 (Shanghai Epizyme Biomedical Technology) was used for antigen retrieval. Slides were subsequently blocked with Antibody Diluent (BioGenex Laboratories, Fremont, CA, USA) and incubated with primary antibodies for 30 min at room temperature. Primary antibodies included ARID1A (1:300), CD8 (1:100, C8/1444B, Dako), GB (1:300), PD‐L1 (1:100) and NPM1 (1:200). After washing with TBST, the slides were incubated with horseradish peroxidase‐linked secondary antibodies (Life Technologies, Carlsbad, CA, USA). Afterwards, tyramide‐conjugated fluorophores (Opal, PerkinElmer, Waltham, MA, USA) were added at a 1:50 dilution and incubated for 10 min at room temperature. This process was repeated for all 5 antibodies, and 4’,6‐diamidino‐2’‐phenylindole (Life Technologies) was diluted at 1:500. Finally, slides were cover slipped with VECTASHIELD Hard Mount (Vector Laboratories, Burlingame, CA, USA). Scanning was performed with a Vectra automated multispectral microscope (PerkinElmer), and inForm software (PerkinElmer) was used for analysis.
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6

Immunofluorescence Imaging of P2X3R, TRPV1 in DRG, TG, and Skin

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Mice were deeply anesthetized by intraperitoneal injection of pentobarbital (100 mg/kg) and perfused transcardially with 20 mL of PBS, followed by 50 mL ice-cold 4% paraformaldehyde. The third to fifth cervical segments of the DRG, TG, or skin were removed, postfixed in the same fixative at 4°C, and placed in 30% sucrose solution for 48 hours at 4°C. DRG, TG (15 μm), or skin (30 μm) sections were incubated in blocking solution (3% normal goat or donkey serum) for 2 hours at room temperature and then incubated for 48 hours (DRG and TG) or 120 hours (skin) at 4°C with primary antibodies: anti-P2X3R (rabbit polyclonal, 1:4000; Chemicon (Temecula, Calif); guinea pig polyclonal, 1:12000; Neuromics, Edina, Minn), anti-GFP (chicken polyclonal, 1:20000; Aves Labs, Tigard, Ore), anti-RFP (rabbit polyclonal, 1:5000; MBL, Nagoya, Japan), and anti-TRPV1 (goat polyclonal, 1:2000; Neuromics). Following incubation, tissue sections were washed and incubated for 3 hours at room temperature in secondary antibody solution (Alexa Fluor 405, 488, or 546, 1:1000). The tissue sections were washed, slide mounted, and subsequently coverslipped with Vectashield Hardmount with 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, Calif). Immunofluorescence images were obtained with confocal laser microscope (LSM510 or LSM700, Carl Zeiss, Thornwood, NY).
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