The slide scanner allows for analysis of entire pancreatic sections and the use of only four different fluorophore combinations. After the acquisition of the tetramer, CD8, and CD45RO staining combination, tissue sections were incubated with 3% H2O2 for 1 hour at room temperature, to inactivate fluorophores chemically, as described previously (42 (link)). Subsequently, the same tissue section was restained using guinea pig anti-insulin (1/1000; Dako), rabbit antiglucagon (1/1000: clone EP3070, Abcam), and mouse anti–HLA-ABC (1/100; clone W6/32, IgG2a, Dako) antibodies. The secondary antibodies were incubated for 1 hour at room temperature and used as follows: anti-rabbit AF555 (1/1000; Invitrogen), anti–guinea pig AF488 (1/500, Jackson ImmunoResearch Laboratories Inc.), and anti-mouse AF647 (1/1000; Thermo Fisher Scientific). After washing and mounting, tissue sections were scanned using a Zeiss AxioScan.Z1 slide scanner (×20 magnification). This approach is essential for in situ studies to visualize the exact location of such autoreactive T cells in the pancreas relative to the islets (fig. S1).
Anti mouse af647
Manufactured by Thermo Fisher Scientific
Anti-mouse AF647 is a fluorescently labeled secondary antibody that binds to mouse primary antibodies. It is designed for use in various immunoassay applications, such as flow cytometry, immunofluorescence, and Western blotting. The AF647 fluorophore emits in the far-red region of the visible spectrum, providing a distinct signal that can be easily detected and distinguished from other fluorescent probes.
Automatically generated - may contain errors
Lab products found in correlation
Microm hm550 cryostat, by Thermo Fisher Scientific (6 mentions)
Anti rabbit af647, by Thermo Fisher Scientific (2 mentions)
Lsm880 fast airyscan confocal microscope, by Zeiss (2 mentions)
Prolong diamond anti fade reagent with dapi, by Thermo Fisher Scientific (2 mentions)
Ep3070, by Abcam (1 mentions)
Anti mouse hrp, by Abcam (1 mentions)
Lysotracker deep red, by Thermo Fisher Scientific (1 mentions)
α tubulin, by Merck Group (1 mentions)
Anti rabbit hrp, by Thermo Fisher Scientific (1 mentions)
Atp5a, by Abcam (1 mentions)
Actin, by Abcam (1 mentions)
Hyqtase, by Thermo Fisher Scientific (1 mentions)
Anti ceacam 1 clone asl 32, by BioLegend (1 mentions)
Anti pd l1 clone 29e 2a3, by BioLegend (1 mentions)
Anti fat1, by Merck Group (1 mentions)
Tryple express, by Thermo Fisher Scientific (1 mentions)
W6 32, by BD (1 mentions)
Accuri c6 cytometer, by BD (1 mentions)
6 protocols using anti mouse af647
1
Multicolor Pancreatic Tissue Analysis
2
Multicolor Imaging and Immunoblotting Antibodies
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The following antibodies and dyes were used: ATP5A (catalog number ab14748, RRID:AB_301447; 1:500; Abcam), Actin (catalog number MAB1501, RRID:AB_2223041; 1:5,000; Millipore), GFP (catalog number ab290, RRID:AB_303395; 1:2,000; Abcam), DsRed (catalog number 632496, RRID:AB_10013483; 1:1,000; Clontech Laboratories), Keima-Red (catalog number M126-3, RRID:AB_10210643; 1:1,000; MBL International), α-Tubulin (catalog number T6793, RRID:AB_477585; 1:5,000; Sigma-Aldrich), and 500 nM LysoTracker Deep Red (L12492 ; Invitrogen). The following secondary antibodies were used for immunoblotting: anti-rabbit HRP (catalog number G-21234, RRID:AB_2536530; 1:10,000; Thermo Fisher Scientific), anti-mouse HRP (catalog number ab67891, RRID:AB_1140250; 1:10,000; Abcam). The following secondary antibody were used for immunofluorescence: anti-mouse AF647 (catalog number A-21235, RRID:AB_2535804; 1:200; Thermo Fisher Scientific).
3
HCMV Infection and Surface Marker Analysis
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HCMV Merlin-infected 6-well dishes of HFFFs were detached with TrypLE Express (Invitrogen) or HyQTase (Thermo), washed in PBS, and resuspended in PBS + 2% fetal calf serum. Cells were incubated with primary antibody for 20–30 min on ice, followed by anti-mouse AF647 (Molecular Probes), and fixed with 1% paraformaldehyde. The following antibodies were used: anti-PD-L1 (clone 29E.2A3, Biolegend), anti-PD-L2 (clone MIH18, Biolegend), anti-CD276 (clone DCN.70, Biolegend), anti-CEACAM1 (clone ASL-32, Biolegend), anti-CD99 (clone HCD99, Biolegend), anti-Cadherin 11 (clone 667034, R and D systems), anti-MHC class I (clone W6-32, Serotec), anti-PCDHGC3 (cat no. ab89520, Abcam), anti-FAT1 (cat no. HPA023882, Sigma) and anti-ROR1 (clone 2H6, Abcam). Samples were acquired with a FACS Accuri flow cytometer and analyzed with FlowJo software (Tree Star).
For intracellular flow cytometry of HCMV IE1, infected cells were trypsinised 24h after infection, fixed in 4% paraformaldehyde then permeabilised using Triton X-100. Fixed cells were incubated with BSA/human serum for 30 min, followed by anti-iE1 antibody (Thermo MA1-7596) for 30 min then anti-mouse AF647.
For intracellular flow cytometry of HCMV IE1, infected cells were trypsinised 24h after infection, fixed in 4% paraformaldehyde then permeabilised using Triton X-100. Fixed cells were incubated with BSA/human serum for 30 min, followed by anti-iE1 antibody (Thermo MA1-7596) for 30 min then anti-mouse AF647.
4
Immunostaining of Cryosectioned Livers
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Dissected livers were fixed in 4% PFA overnight at 4°C and washed with PBS/0.1% Tween 20 before incubation in 30% sucrose in PBS overnight at 4°C. Livers were aligned in a tissue mould, embedded in OCT and frozen on dry ice. The livers were sectioned at 10µm intervals using a Thermo Fisher Scientific Microm HM550 cryostat. Sections were washed with PBS before blocking with 10% FCS in PBS/0.3% Triton X-100. Incubation with primary antibodies was performed at 4°C overnight, while incubation with secondary antibodies was performed at room temperature for 1 hr. Antibodies used in this work were: 1:2000 a-Tubulin DM1A (CST, #3873), 1:1000 γ-H2AX (gift of James Amatruda), 1:250 cleaved caspase-3 (CST, #9664), 1:500 anti-rabbit AF647 (Thermo Fisher Scientific, #A31573) and 1:500 anti-mouse AF647 (Thermo Fisher, #A21235). Prolong Diamond Antifade reagent with DAPI (Thermo Fisher #P36962) was used for slide mounting. A Zeiss LSM880 Fast Airyscan Confocal microscope with a ×63 objective was used for image acquisition and ImageJ for image analysis.
5
Flow Cytometry Analysis of Cell Markers
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Cells were dissociated with TrypLE, stained with antibodies targeting HLA (W632), PDGFRα (BD Pharmingen) or CD155 (D171; Invitrogen) followed by anti-mouse AF647 (Thermo), before being washed and fixed in 4% PFA, then run on a Accuri C6 cytometer (BD) and analysed in FlowJo. 'All gates were set using a non-binding isotype matched control antibody (for the negative gate), and cells that had been transduced with a control RAd vector lacking expression of Cas9 (for the positive gate).
6
Liver Histology and Immunostaining
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Dissected livers were fixed in 4% PFA overnight at 4 o C and washed with PBS/0.1% Tween 20 before incubation in 30% sucrose in PBS overnight at 4 o C. Livers were aligned in a tissue mould, embedded in OCT and frozen on dry ice. The livers were sectioned at 10 µm intervals using a Thermofisher Scientific Microm HM550 cryostat. Sections were washed with PBS before blocking with 10% FCS in PBS/0.3% Triton X-100. Incubation with primary antibodies was performed at 4°C overnight, while incubation with secondary antibodies was performed at room temperature for 1 h. Antibodies used in this work were: 1:2000 a-Tubulin DM1A (CST, #3873), 1:1000 γ-H2AX (gift of James Amatruda), 1:250 cleaved caspase 3 (CST, #9664), 1:500 anti-rabbit AF647 (Thermofisher scientific, #A31573) and 1:500 anti-mouse AF647 (Thermofisher, #A21235). Prolong Diamond Antifade reagent with DAPI (Thermofisher #P36962) was used for slide mounting. A Zeiss LSM880 Fast Airyscan Confocal microscope with a 63x objective was used for image acquisition and ImageJ for image analysis.
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