Islet graft-bearing kidneys, pancreases, and intestines fixed in 4% paraformaldehyde were embedded in optimal cutting temperature compound and frozen on dry ice. 6–10μm sections were made on a Leica CM3050 S (Leica Biosystems, Buffalo Grove, IL). Immunofluorescent staining was performed using standard methods. Briefly, sections were blocked in 5% BSA for 1hr then incubated with primary antibodies overnight at 4°C. Sections are washed 3 × 5 min before incubation with secondary antibodies for 2 hours at room temperature and washed 3 × 5 min again. Slide covers were secured with Hard-set Mounting Medium with DAPI (Santa Cruz Biotechnology, Dallas TX). Slides were imaged on an EVOS M5000 Cell Imaging System (ThermoFisher). Post-processing and color channel merging was performed in Fiji (http://fiji.sc/ ) (Schindelin et al., 2012 (link)). Primary antibodies (1:100–200): αCD3 (17A2) and αCD45 (30-F11) were purchased from BioLegend, insulin (a0564) from Dako (Carpinteria, CA). Secondary antibodies (1:200–500): CF-594 and CF-488A α-Guinea Pig were purchased from MilliporeSigma (St. Louis, MO), Alexa Fluor-594 and Alexa Fluor-488 α-Rat, and Alexa Fluor-594 α-Rabbit were purchased from BioLegend.
αcd45 30 f11
Manufactured by BioLegend
αCD45 (30-F11) is a monoclonal antibody that recognizes the CD45 antigen, also known as the Leukocyte Common Antigen (LCA). CD45 is a transmembrane protein tyrosine phosphatase that is expressed on the surface of all hematopoietic cells, including T cells, B cells, monocytes, and granulocytes. The 30-F11 clone is commonly used for the identification and analysis of mouse CD45-positive cells.
Automatically generated - may contain errors
Lab products found in correlation
Evos m5000 cell imaging system, by Thermo Fisher Scientific (3 mentions)
αcd3 17a2, by BioLegend (3 mentions)
Cf 594, by Merck Group (3 mentions)
Alexa fluor 488 α rat, by BioLegend (3 mentions)
Alexa fluor 594 α rabbit, by BioLegend (3 mentions)
Cf 488a α guinea pig, by Merck Group (3 mentions)
Cm3050 s, by Leica (3 mentions)
Alexa fluor 594, by BioLegend (2 mentions)
Hard set mounting medium with dapi, by Santa Cruz Biotechnology (2 mentions)
Insulin a0564, by Agilent Technologies (2 mentions)
Glucagon, by Thermo Fisher Scientific (1 mentions)
Somatostatin, by Agilent Technologies (1 mentions)
Insulin, by BioLegend (1 mentions)
Uea 1, by Merck Group (1 mentions)
M5 114, by Thermo Fisher Scientific (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Anti cd4, by Miltenyi Biotec (1 mentions)
Anti cd8 magnetic beads, by Miltenyi Biotec (1 mentions)
α mhcii, by Thermo Fisher Scientific (1 mentions)
4 protocols using αcd45 30 f11
1
Immunofluorescent Analysis of Islet Grafts
2
Multicolor Immunofluorescent Staining of Islet Grafts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Islet graft and pancreas sections were fixed in 4% paraformaldehyde, embedded in optimal cutting temperature compound, and frozen on dry ice. Embedded grafts were sectioned on a Leica CM3050 S (Leica Biosystems, Buffalo Grove, IL). Standard immunofluorescent staining techniques were used on 6 µm sections. Sections were blocked in 5% BSA for 30 min, incubated with primary antibodies overnight at 4 °C, washed before incubation with secondary antibodies for 2 h at room temperature, and finally washed again. Slide covers were secured with Hard-set Mounting Medium with DAPI (Santa Crus Biotechnology, Dallas TX). Slides were imaged on an EVOS M5000 Cell Imaging System (ThermoFisher) and color channels were merged using Fiji (http://fiji.sc/ ). Primary antibodies (1:100): αCD3 (17A2) and αCD45 (30-F11) were purchased from BioLegend, insulin (Catalog #: a0564) and somatostatin (Catalog #: A0566) from Dako (Carpinteria, CA), glucagon (Catalog #: PA5-88091) from ThermoFisher. Secondary antibodies (1:1000): CF-594 and CF-488A α-Guinea Pig were purchased from MilliporeSigma (St. Louis, MO); Alexa Fluor-594 α-Rat, Alexa Fluor-488 α-Rat, and Alexa Fluor-594 α-Rabbit were purchased from BioLegend.
3
Isolation and Characterization of Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human BLS cell lines and in vitro generated B cell mutants have been described and are established human cell lines [1 (link),30 (link)]. T cells were enriched using anti-CD4 and/or anti-CD8 magnetic beads (Miltenyi Biotec). For all flow cytometric analyses, gating on living cells and exclusion of doublets was performed. Enriched TEC suspensions [2 (link)] were washed in PBS, 2% FCS, 5mM EDTA and stained for flow cytometry using death exclusion markers (either DAPI or 7AAD), UEA1 (Sigma) and the following mAb-conjugated mix: α-CD45 (30F11, BioLegend) and α-BP1 (6C3; BioLegend), α-MHCII (M5/114.15.2; eBioscience) and α-EpCAM (G8.8, Developmental Studies Hybridoma Bank, Iowa), α-H2-Db (B22/24g) and α-H2-Kb (B8.24.3). Splenocytes were preincubated with anti-CD16/32 (2.4G2) to block FcRs and stained using Abs against CD8a (Ly-2), CD3e (145-2C11), CD4 (L3T4), CD11b (M1/70), CD11c (N418), CD19 (1D3), H2-Db (28-14-8), H2-Kb (AF6–88.5.5.3), MHCII (M5/114.15.2), NK1.1 (PK136), B220 (RA3-6B2), Qa-2 (69H1-9-9) (all from eBioscience). Streptavidin conjugated to different fluorophores was from eBioscience. Stainings were performed with appropriate combinations of fluorophores. Data was acquired with a FACSCanto flow cytometer (Becton Dickinson) and analyzed using FlowJo software (Tree Star).
4
Immunofluorescent Analysis of Islet Grafts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Islet graft-bearing kidneys, pancreases, and intestines fixed in 4% paraformaldehyde were embedded in optimal cutting temperature compound and frozen on dry ice. 6–10μm sections were made on a Leica CM3050 S (Leica Biosystems, Buffalo Grove, IL). Immunofluorescent staining was performed using standard methods. Briefly, sections were blocked in 5% BSA for 1hr then incubated with primary antibodies overnight at 4°C. Sections are washed 3 × 5 min before incubation with secondary antibodies for 2 hours at room temperature and washed 3 × 5 min again. Slide covers were secured with Hard-set Mounting Medium with DAPI (Santa Cruz Biotechnology, Dallas TX). Slides were imaged on an EVOS M5000 Cell Imaging System (ThermoFisher). Post-processing and color channel merging was performed in Fiji (http://fiji.sc/ ) (Schindelin et al., 2012 (link)). Primary antibodies (1:100–200): αCD3 (17A2) and αCD45 (30-F11) were purchased from BioLegend, insulin (a0564) from Dako (Carpinteria, CA). Secondary antibodies (1:200–500): CF-594 and CF-488A α-Guinea Pig were purchased from MilliporeSigma (St. Louis, MO), Alexa Fluor-594 and Alexa Fluor-488 α-Rat, and Alexa Fluor-594 α-Rabbit were purchased from BioLegend.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!