Multicolor immunofluorescence analyses were performed using 3-μm-thick sections of formalin-fixed paraffin-embedded (FFPE) tissues. Briefly, slides were deparaffinized in xylene and hydrated in a series of decreasing graded ethanol series. After heat-induced antigen retrieval in citrate buffer (pH = 6), samples were permeabilized with 0.5% Triton X-100, blocked with 5% goat serum-phosphate-buffered saline (PBS), and sequentially co-stained with antibodies recognizing VCAM-1 (Abcam, ab134047), CD248 (Abcam, ab217535), 4-HNE (Abcam, ab46545), FAPα (Invitrogen, BMS168; Abcam, ab218164), Mfap4 (Thermo, PA5-24865), Sparcl1 (Santa Cruz, sc-514275), F4/80 (Cell Signaling Technology, #70076), rabbit IgG Isotype Control (Invitrogen, 31235), mouse IgG Isotype Control (Invitrogen, 14-4714-82), and DAPI. A TSA indirect kit (PerkinElmer) was used according to the manufacturer’s instructions. Vectra® Polaris™ Imaging System (Akoya Biosciences) was used to collect images, and image analysis was performed using HALO™ Image Analysis Software.
Rabbit igg isotype control
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, Japan
The Rabbit IgG isotype control is a laboratory reagent used in flow cytometry and other immunoassays to establish appropriate background signal levels. It serves as a negative control by providing a reference point for non-specific binding of antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Mouse igg isotype control, by Thermo Fisher Scientific (4 mentions)
F4 80, by Cell Signaling Technology (2 mentions)
Tsa indirect kit, by PerkinElmer (2 mentions)
Isotype control mouse igg2a, by BD (2 mentions)
Fitc conjugated goat anti mouse pab, by Agilent Technologies (2 mentions)
Facsdiva v6, by BD (2 mentions)
Cd279 pd 1, by BioLegend (2 mentions)
Tru nuclear transcription buffer set, by BioLegend (2 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (2 mentions)
Anti ha antibody, by Cell Signaling Technology (2 mentions)
Magnetic protein a g beads, by Thermo Fisher Scientific (2 mentions)
Ab170511, by Abcam (2 mentions)
Goat anti human igm μ chain specific conjugated agarose, by Merck Group (2 mentions)
Rat igg2b isotype control, by BioLegend (2 mentions)
Anti asialo gm1, by Fujifilm (2 mentions)
Anti cd200r3 ba103, by Hycult Biotech (2 mentions)
Vectra polaris imaging system, by Akoya Biosciences (1 mentions)
Ab134047, by Abcam (1 mentions)
Ab46545, by Thermo Fisher Scientific (1 mentions)
Streptavidin peroxidase kit, by ZSGB-BIO (1 mentions)
37 protocols using rabbit igg isotype control
1
Multicolor Immunofluorescence of FFPE Tissues
2
Immunohistochemical Analysis of Oxidative Stress
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Immunohistochemical staining was performed using a streptavidin–peroxidase kit (ZSGB-Bio, China). The primary antibodies targeted the following proteins or modifications: 8-OHdG (Abcam, ab48508), 4-HNE (Abcam, ab48506, ab46545), FAPα (Abcam, ab53066), GPX4 (Abcam, ab125066), GCLC (Abcam, ab53179), GCLM (Abcam, ab126704), p-NF-κB p65 (Thermo Fisher, 44-711G), rabbit IgG Isotype Control (Invitrogen, 31235), and mouse IgG Isotype Control (Invitrogen, 14-4714-82) with a standard avidin-biotin HRP detection system according to the instructions of the manufacturer (anti-mouse/rabbit HRP-DAB Cell & Tissue Staining Kit, R&D Systems, Minneapolis, MN). Tissues were counterstained with hematoxylin, dehydrated, and mounted. Quantitative analysis was performed with Image-Pro Plus Version 6.0 Software or HALO™ Image Analysis Software. For quantitative analysis using Image-Pro Plus, the percentage of positive cells was graded on a scale of 0 to 4 (0: negative, 1: 0–25%, 2: 26–50%, 3: 51–75%, 4: 76–100%). The staining intensity was further quantified from 0 to 3 (1: weak, 2: moderate, 3: intensive). The final score was obtained by multiplying the staining intensity score and percentage score.
3
Survivin Neutralization Assay
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Survivin neutralization was performed by adding 20 μg/ml of an anti-survivin antibody (ab76424; Abcam, Milton, Cambridge, UK) for 1 h at room temperature before adding the medium to THP-1 or HepG2 cells. A negative epitope control (Rabbit IgG Isotype Control, Invitrogen) was included in each experiment.
4
Immunohistochemical Analysis of SAP, CRP, and C1q
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Rabbit anti-human SAP polyclonal antibody (pAb) (565191, Calbiochem), rabbit anti-human CRP pAb (235752, Calbiochem), rabbit anti-human C1q pAb (A0136, Dako), mouse anti-human complement component C5b-9 monoclonal antibody (mAb) (IgG2a) (011-01, Antibody Shop), mouse IgG1 isotype control (BD Biosciences), mouse IgG2a isotype control (BD Biosciences), rabbit IgG isotype control (Invitrogen), FITC-conjugated goat anti-rabbit pAb (F1262, Sigma-Aldrich), FITC-conjugated goat anti-mouse pAb (F0479, Dako), mouse anti-human CD45 FITC/CD14 PE (342408, BD Biosciences), mouse anti-human CD11b APC-Cy7 (560914, BD Biosciences), Alexa-555-conjugated goat anti-mouse IgG (A-21424, Thermo Fischer), biotinylated goat anti-rabbit IgG (BA-1000, Vector Laboratories), or biotinylated goat anti-mouse IgG (BA-9200, Vector Laboratories) are the commercial antibodies.
5
Immunohistochemical Analysis of IL-17RA in Mouse Lung
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Immunohistochemical staining was performed with mouse lung sections using a commercial detection system (TP-015-HA, Thermo Scientific) as previously described [55 (link)]. Heat-induced epitope retrieval was performed by placing the slides in a glass slide container with citrate buffer (0.1 M citric acid and 0.1 M sodium citrate, pH 6.0) in a water bath at 95°C for 20 min. After cooling to room temperature, the sections were blocked by serial incubation with hydrogen peroxide, Ultra V block, and 5% goat serum in PBS. The blocked lung sections were incubated with anti-IL-17RA Ab (PA5-34571, Invitrogen) or rabbit IgG isotype control (08–6199, Invitrogen) overnight at 4°C in a humidified chamber. After incubation with biotinylated goat anti-mouse IgG and streptavidin-HRP, the sections were rinsed with PBS and incubated with 3-amino-9-ethylcarbazole, a chromogenic HRP substrate, followed by counterstaining with hematoxylin.
6
Multiparametric Immunophenotyping of PBMCs
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PBMCs were isolated as described above and stained with the following fluorochrome-conjugated antibodies: CD19, CD27, CD38, IgD (BD Biosciences), CD11c, CD3, CXCR5, and CD279 (PD-1) (BioLegend) in the presence of 10% normal mouse serum. For intracellular staining, cells were fixed, permeabilized and stained using the Tru-nuclear Transcription Buffer set (BioLegend). Cells were blocked with 50 μg/ml Rabbit IgG and stained with 2 μg/ml rabbit anti-human ATF3 (USBIO) or Rabbit IgG isotype control (Invitrogen). Single color stained anti-mouse Ig beads (Bangs) were used as compensation controls. Flow cytometry were collected with BD FACSDiva v6.2 and data analysis was performed FlowJo (TreeStar) v10.4.
7
Antibody Characterization for SCARB2 Analysis
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The mouse anti-CD162 (PSGL-1) mAb (clone KPL-1, Cat# 556052, Lot# 3225805) were purchased from BD Biosciences. The mouse IgG1 isotype control (clone MOPC-21, Cat# 400124, Lot# B136629) and IgG2a (clone MOPC-173, Cat# 400202, Lot# B223740) were purchased from BioLegend. The goat anti-SCARB2 pAb (Cat# AF1966, Lot# KKY0115011 [Figs 5A, 5B and 8A ], KKY0118021 [S1 and S7 Figs], KKY0118031 [Figs 8B , 9B , S2B and S6 ], KKY0121011 [Figs 2A , 3 and 9C ]), normal goat IgG (Cat# AB-108-C, Lot# ES4119121, except for Fig 9C where Lot# ES4521041 was used), and the goat anti-human IgG Fc Ab (Cat# G-102-C, Lot# WBT1519101) were purchased from R&D Systems. The rabbit anti-SCARB2 mAb (clone 12H5L1, Cat# 702770, Lot# 2110715; clone 22H6L14, Cat# 703037, Lot# 2360697), rabbit IgG isotype control (Cat# 10500C, Lot# UA276761), the mouse anti-CD63 mAb (clone MEM-259, Cat# MA1-19281, Lot# WG3317432A), and the mouse anti-CD107a (LAMP-1) mAb (clone eBioH4A3, Cat# 14-1079-80, Lot# 2440973) were purchased from Invitrogen. The mouse anti-EEA1 mAb (clone 3C10, Cat# M176-3MS, Lot# 003) was purchased from MBL.
8
Isolation and Analysis of Pancreatic Leukocytes
9
Immunohistochemical Detection of CCDC38
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Fixed lung tissue was sectioned and mounted. Slides were treated in Histo-Clear and then re-hydrated using 100% ethanol and 95% ethanol washes. Antigen retrieval was carried out by steaming the tissue samples for 30 minutes in sodium citrate buffer (2.1 g Citric Acid [Fisons - C-6200-53]+13 ml 2M NaOH [Fisher - S-4880/53] in 87 ml H2O). Tissue was then treated with peroxidise blocking solution (Dako - S2023), followed by treatments with a 1 in 50 dilution of rabbit anti-CCDC38 antibody (Sigma HPA039305; 0.2 mg/ml) or a 1 in 50 dilution of the Rabbit IgG Isotype control (Invitrogen 10500C, diluted to 0.2 mg/ml). Secondary antibody staining and DAB treatment was carried out using the EnVision Detection Systems Peroxidase/DAB, Rabbit/Mouse kit (Dako – K5007). Tissue was then counterstained with Mayers Hematoxylin solution (Sigma – 51275) before being dehydrated using 95% ethanol and 100% ethanol washes. Slides were mounted using Vectamount (Vector Laboratories - H-5000).
10
Glucocorticoid Receptor Chromatin Binding Assay
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We analysed the sequence for the rx3 gene (ENSDARG00000052893, chromosome:GRCz11:21:10755554:10759823:1). Primers for rx3 GRE1 and GRE2 are described in supplementary data 2 and were designed using Primer3. GR bounded chromatin was prepared following the protocol from Idilli et al.88 (link), with minor modifications. For each sample, 100 dissected 5 dpf larval heads (without eyes) were incubated for 10 min at room temperature in 1% formaldehyde in PBS with protease inhibitors (A32955, Thermo Scientific) for cross-linking. We used a glucocorticoid receptor polyclonal antibody (24050-1-AP, Proteintech) and Rabbit IgG Isotype Control (10500 C, Invitrogen) as primary antibodies. For each sample, 3 technical replicates were run on the PCR machine.
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