Formalin-fixed paraffin-embedded (FFPE) tissues were stained with Vectastain Elite ABC-HRP rat kit (PK-6104; Vector Laboratories) using rat anti–Clever-1 (clone S2–7/H7) as previously described (24 (link)). CD8 and CD163 staining was performed at Covance according to their validated protocols. For immunofluorescence (IF) analysis, rabbit anti-Granzyme B (PA1–26616; Invitrogen), mouse anti-CD8 (LS-C311966; LSbio), or irrelevant isotype controls and the secondary antibodies anti–rabbit-AF488 (A11034; Life Technologies) and anti–mouse-AF546 (A11030; Invitrogen) were used. The slides were mounted with Prolong Gold containing DAPI (Thermo Fisher).
Anti rabbit af488
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Anti-rabbit AF488 is a fluorescently-labeled secondary antibody that specifically binds to rabbit primary antibodies. The AF488 fluorophore attached to the secondary antibody emits green fluorescence when excited by a compatible light source, allowing for the detection and visualization of rabbit antigens in various applications.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Streptavidin af555, by Thermo Fisher Scientific (3 mentions)
Avidin biotin blocking kit, by Vector Laboratories (3 mentions)
Anti mouse af546, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Anti rabbit af568, by Thermo Fisher Scientific (2 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (2 mentions)
Ebioscience permeabilization buffer, by Thermo Fisher Scientific (2 mentions)
Antibody rabbit anti ttr, by Abcam (2 mentions)
Avidin biotin blocking reagent, by Vector Laboratories (2 mentions)
Anti gfp af488, by Thermo Fisher Scientific (2 mentions)
Anti cd45.2 biotin 104, by Thermo Fisher Scientific (2 mentions)
Anti cd90.1 apc his51, by Thermo Fisher Scientific (2 mentions)
Anti laminin l9393, by Abcam (2 mentions)
Anti epcam af647 buv421 g8.8, by BioLegend (2 mentions)
Oct media, by Sakura Finetek (2 mentions)
Cd11c biotin, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (2 mentions)
Tryamide blocking kit, by PerkinElmer (2 mentions)
Anti f4 80, by Abcam (2 mentions)
18 protocols using anti rabbit af488
1
Immunohistochemical Analysis of Clever-1, CD8, and CD163
2
Characterization of zDHHC15 Protein Expression
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Primary antibodies used were: anti-zDHHC15 (Abcam, Cambridge, MA, #ab121203, 1:250, western blot), anti-zDHHC15 (Thermo Scientific, Waltham, MA, #PA39327, immunofluorescence, 1:100), anti-Flag (Sigma Aldrich, St Louis, MO, #F3165, 1:1000), anti-Myc (Origene, Rockville, MD, #TA150121, 1:1000), anti-β-actin (Novus Biologicals, Centennial, CO, #NB600-503, 1:1000), anti-PSD-95 (Abcam, Cambridge, MA, #ab2723, 1:500), anti-gephyrin (Synaptic Systems, Göttingen, Germany #147011, 1:500), anti-GAD65 (Synaptic Systems, Göttingen, Germany #198104, 1:500), anti-GFAP (Synaptic Systems, Göttingen, Germany #173004, 1:400), anti-MAP2 (Millipore, Burlington, MA, MB3418, 1:2000), anti-giantin (Synaptic Systems, Göttingen, Germany, #263004, 1:500), anti-GM130 (BD Biosciences, Franklin Lakes, NJ, #610822, 1:200). Secondary antibodies from Life Technologies (Carlsbad, CA,) used: (1:500), anti-mouse AF568 IgG2a (#A21134), anti-mouse AF647 IgG1 (#21240), anti-rabbit AF488 (#A11008), anti-rabbit AF568 (#A11011), anti-guinea pig AF633 (#A21105). HRP conjugated secondary antibodies were obtained from Bio-Rad (Hercules, CA): goat anti-mouse #170-6516 and goat anti-rabbit (#170-6515, 1:300). DAPI was used for nuclear staining (Life Technologies, Carlsbad, CA, #D1306, 1:1000).
3
Multicolor Flow Cytometry Analysis
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Cells were resuspended in FACS buffer, and stained as previously described [39 (link), 40 (link)]. In short, cells were incubated with an anti-mouse CD16/32 unconjugated antibody (clone 93, BioLegend) and stained with fixable viability dye Zombie NIR (BioLegend), for 20 min at 4 °C. Subsequently, cells were stained (30 min, 4 °C) using fluorochrome-conjugated antibodies. CD11b (PerCP-Cy5.5, clone M1/70) purchased from eBioscience, CD45 (BV510, clone 30-F11), MHCII (BV711, clone M5/114.15.2), VE-cadherin (BV421, clone BV13), and gp38 (AF488, clone PMab-1) purchased from BioLegend. CD31 (APC, clone MEC13.3) purchased from BD Biosciences. Cells were washed (400 × g, 5 min 4 °C) with FACS buffer, fixed in 4% PFA for 15 min at 4 °C and re-suspended in FACS buffer. For CP, cells were additionally permeabilized with eBioscience™ Permeabilization buffer (Invitrogen), incubated with antibody rabbit-anti-TTR (Abcam) for 40 min, and stained with secondary antibody anti-rabbit AF488 (ThermoFisher). Cells were acquired using Attune NxT Flow Cytometer (ThermoFischer). Data were analyzed using FlowJo software (version 10.5.3, FlowJo LLC, OR, USA).
4
Visualizing Intestinal Lymphocyte Localization
5
Mitochondrial Staining and Immunofluorescence
6
Visualizing Intestinal Lymphocyte Localization
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The ileum was harvested at day 9 p.i., fixed in 2% paraformaldehyde, rehydrated in 20% sucrose in PBS, and flash frozen in optimal cutting temperature (OCT) media (Sakura Finetek, Torrance, CA, USA). The tissue sections were cut at a thickness of 7–8 μm and treated with ice cold acetone before storage at −80°C. Tissue sections were treated with avidin-biotin blocking reagents (Vector Laboratories, Newark, CA, USA) and stained with the following reagents: anti-CD45.2-biotin (104; Invitrogen), anti-CD90.1-APC (HIS51; Invitrogen), anti-laminin (L9393; Abcam, Cambridge, United Kingdom), anti-GFP-AF488 (Thermo Fisher Scientific), and anti-EpCam-AF647/BUV421 (G8.8; Biolegend), followed by Streptavidin-AF555 (Thermo Fisher Scientific), anti-rabbit AF488 (Thermo Fisher Scientific), and 1 μg/ml DAPI (Millipore Sigma). Stained slides were mounted with Prolong Gold antifade reagent, imaged using a Keyence fluorescence BZ-X series microscope (Osaka, Osaka, Japan), and analyzed using the Adobe Photoshop software. For the quantification of cells in lymphocyte clusters, the cells were counted in multiple sections for each mouse for > 700 total cells/mouse and > 200 cells in lymphocyte clusters/mouse. For examining the distance of LP Trm cells from the epithelium, the distance between the surface of each cell and the base of the nearest epithelial cell was determined for 150–360 cells/mouse.
7
Immunohistochemistry of Myeloid Cells in Neuroinflammation
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Immunohistochemistry was performed with co-cultures of organotypic hippocampal slices from CX3CR1GFP mice with 2D2 T cells and spinal cord sections of EAE diseased CX3CR1.GFP animals. Iba-1+/CX3CR1+ myeloid cells were stained with anti-mouse GFP (polyclonal-rabbit; Abcam; 270F3 mouse; Synaptic Systems) or anti-mouse Iba-1 (polyclonal-rabbit; Abcam) and anti-rabbit-AF488 (polyclonal; Thermo Fisher Scientific). CD4 was stained with anti-mouse CD4-AF647 (RM4-5 rat; BD Biosciences). MHC-II was stained with anti-mouse MHC-II (2G9 rat; BD Biosciences), CD206 with anti-mouse CD206 (C068C2 rat; BioLegend) and anti-rat-AF568 (polyclonal, Thermo Fisher Scientific), CD31 with anti-mouse CD31 (polyclonal-rabbit, Thermo Fisher Scientific) and anti-rabbit-AF647 (polyclonal; Thermo Fisher Scientific). The cell nucleus was stained with DAPI (Thermo Fisher Scientific).
8
Comprehensive Immunophenotyping Approach
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Anti-HSP90 (clone 3H3C27), anti-SHIP1 (clone PICI-A5), FITC-conjugated rat anti-mouse GR1 (clone RB6-8C5), PE-conjugated rat anti-mouse/human CD11b (clone M1/70), PE/Cy7-conjugated rat anti mouse/human CD45 (clone 30-F11), PerCP/Cy5.5-conjugated rat anti F4/80 (clone BM8), APC-conjugated rat anti-B220 (clone RA3-6B2), PE/Cy7-conjugated rat anti CD3 (clone 17A2) and pacific blue-conjugated rat anti-LY6G (clone 1A8) were from BioLegend (London, UK); anti-PTEN (clone D4.3), anti-PKB (clone 11E7), anti-PKB T308 (clone C25E6) and anti-PKB S473 (clone D9E) were from Cell Signaling Technology (London, UK). Rabbit IgG (I8140) was obtained from Sigma. Anti-SHIP2 (AF5389) and PE-conjugated rat anti-CD64 (clone FAB20741P) were from R&D Systems (Abingdon, UK) and biotinylated anti-PI(3,4)P2 (z-B034) was from Echelon Biosciences (Salt Lake City, UT, USA); streptavidin-AF647, AF488-conjugated phalloidin, AF568-conjugated phalloidin, and secondary antibodies anti-rat AF488, anti-rabbit AF568 and anti-rabbit AF488 were obtained from Thermo Fisher Scientific (Loughborough, UK).
9
Multiparametric Flow Cytometry Analysis
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Cells were resuspended in FACS buffer, and stained as previously described (39, 40) .
All antibodies were purchased from BioLegend unless otherwise stated. In short, cells were incubated with an anti-mouse CD16/32 antibody (clone 93, BioLegend, 101302) and stained with fixable viability dye ZombieNIR, for 20 min at 4°C. Subsequently, cells were stained (30 min, 4 °C) using fluorochrome-conjugated antibodies CD11b (PerCP-Cy5.5) eBioscience, CD45 (BV510), MHCII (BV711), and CD31 (APC) BD Biosciences. Cells were washed (400 g, 5 min 4°C) with FACS buffer, fixed in 4% PFA for 15 min at 4 °C and re-suspended in FACS buffer. For CP, cells were additionally permeabilized with eBioscience™ Permeabilization Buffer (Invitrogen), incubated with antibody rabbit-anti-TTR (Abcam) for 40 min, and stained with secondary antibody anti-rabbit AF488 (ThermoFisher). Cells were acquired using Attune NxT Flow Cytometer (Thermo Fischer). Data was analysed using FlowJo software (version 10.5.3, FlowJo LLC, OR, USA).
All antibodies were purchased from BioLegend unless otherwise stated. In short, cells were incubated with an anti-mouse CD16/32 antibody (clone 93, BioLegend, 101302) and stained with fixable viability dye ZombieNIR, for 20 min at 4°C. Subsequently, cells were stained (30 min, 4 °C) using fluorochrome-conjugated antibodies CD11b (PerCP-Cy5.5) eBioscience, CD45 (BV510), MHCII (BV711), and CD31 (APC) BD Biosciences. Cells were washed (400 g, 5 min 4°C) with FACS buffer, fixed in 4% PFA for 15 min at 4 °C and re-suspended in FACS buffer. For CP, cells were additionally permeabilized with eBioscience™ Permeabilization Buffer (Invitrogen), incubated with antibody rabbit-anti-TTR (Abcam) for 40 min, and stained with secondary antibody anti-rabbit AF488 (ThermoFisher). Cells were acquired using Attune NxT Flow Cytometer (Thermo Fischer). Data was analysed using FlowJo software (version 10.5.3, FlowJo LLC, OR, USA).
10
Immunostaining of Thymic Tissue Sections
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Freshly isolated thymic lobes were frozen in optimal cutting temperature (OCT) compound (Tissue-Tek) and cryosectioned at a thickness of 10 μm. Tissue sections were fixed with ice-cold acetone for 5 min and blocked with the Avidin/Biotin Blocking Kit (Vector Laboratories) and Protein Block (Dako) according to the manufacturer’s protocol. Tissue sections were then incubated with primary antibodies at 4°C overnight: rabbit anti-mouse CD26 (DPP4) [EPR5883(2), Abcam] and biotin anti-mouse podoplanin (8.1.1, BioLegend). Secondary antibody staining was performed at RT for 30 min with anti-rabbit:AF488 (Invitrogen) and streptavidin-AF555 (Invitrogen). Nuclei were stained with Hoechst 34580 in PBS (according to the manufacturer’s protocol). Sections were mounted with ProLong Gold Antifade Mountant (Thermo Fisher Scientific) and acquired using an LSM700 confocal microscope (Carl Zeiss AG). Image analysis was performed with ImageJ software (W. S. Rasband, ImageJ, U.S. National Institutes of Health, Bethesda, MD).
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