For immunofluorescence staining, cryosections of the spleens from SRBCs immunized NSD1B WT and NSD1B KO mice were fixed with ice-cold acetone for 10 mins at room temperature. Then the sections were incubated with 2% BSA for 2 hr at room temperature. After that, sections were incubated with Gl7-FITC (BioLegend) and IgD-PE (BioLegend) for 2 hr at room temperature then incubated with DAPI for 5 mins at room temperature. Images were captured using OLYMPUS BX53 microscope.
Igd pe
Manufactured by BioLegend
Sourced in United States
IgD-PE is a fluorescent-labeled antibody that binds to the immunoglobulin D (IgD) antigen. It can be used for the identification and analysis of IgD-expressing cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Tissue tek oct compound, by Sakura Finetek (2 mentions)
Gl7 af488, by BioLegend (2 mentions)
Gl7 fitc, by BioLegend (1 mentions)
Cd24 pe cy7, by BioLegend (1 mentions)
Cd21 fitc, by BioLegend (1 mentions)
Macsquant analyzer, by Miltenyi Biotec (1 mentions)
Cd19 pacific blue, by BioLegend (1 mentions)
Igm fitc, by BD (1 mentions)
Cd27 apc cy7, by BioLegend (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Cd25 pe, by BioLegend (1 mentions)
B220 apc cy7, by BioLegend (1 mentions)
F4 80 pe, by BioLegend (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Brefeldin a, by BioLegend (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
F4 80 percp cy5, by BioLegend (1 mentions)
Tnf α pe, by BioLegend (1 mentions)
Cd19 percp cy5, by BioLegend (1 mentions)
Cd45.2 apc, by BioLegend (1 mentions)
7 protocols using igd pe
1
Immunofluorescence Analysis of Germinal Centers
2
Multiparametric Flow Cytometry Analysis
3
Comprehensive Immune Cell Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We used the following gating schemes: total leukocytes (CD45.2+), total B cells (CD19+ CD3−), B-2 (CD19+ CD3− B220hi CD5− CD23+), B-1a (CD19+ CD3− IgM+ IgD− B220lo CD5+ CD23−), B-1b (CD19+ CD3− IgM+ IgD− B220lo CD5− CD23−), regulatory B [Breg] cells (CD19+ B220+ CD22+ CD5− IgM+ IgD+), total T cells (CD19− CD3+), Treg cells (CD19− CD3+ CD4+ CD25+), and macrophages (CD19− CD3− CD11b+ F4/80+). Dead cells were distinguished by Live/Dead Fixable Aqua staining (Life Technologies). Baselines for IL-10 EGFP mice were set using age- and diet-matched C57BL/6J mice. For macrophage intracellular cytokine staining, cells were stimulated with LPS (1 μg/mL; Sigma-Aldrich) and brefeldin A (5 μg/mL; BioLegend) overnight and stained using the Cytofix/Cytoperm Kit (BD Biosciences) according to the vendors’ instructions. Data were acquired on an LSR II flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star).
CD16/32, CD3-PacBlue, CD5-PE-Cy5, CD19-PerCP-Cy5.5, CD22-PE, CD23-PE-Cy7, CD25-PE, CD45.2-APC, B220-APC-Cy7, F4/80-PE, F4/80-PErCP-Cy5.5, IgD-PE, and TNF-α–PE antibodies were from BioLegend. IgM-efluor650, CD4-efluor650, and CD11b-efluor605 antibodies were from eBioscience.
CD16/32, CD3-PacBlue, CD5-PE-Cy5, CD19-PerCP-Cy5.5, CD22-PE, CD23-PE-Cy7, CD25-PE, CD45.2-APC, B220-APC-Cy7, F4/80-PE, F4/80-PErCP-Cy5.5, IgD-PE, and TNF-α–PE antibodies were from BioLegend. IgM-efluor650, CD4-efluor650, and CD11b-efluor605 antibodies were from eBioscience.
4
Multi-Marker Immunofluorescence of Frozen Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Kidneys and spleens were embedded in Tissue-Tek OCT Compound (Sakura Finetek, Torrance, CA) and rapidly frozen in a dry ice and 2-methylbutane freezing bath. The frozen OCT-embedded samples were cryosectioned, and unstained slides were stored at -80°C. Subsequently, these frozen slides were allowed to reach room temperature and dry for 30 minutes before fixation in -20°C cold acetone for 10 minutes. After a wash in PBS, the slides were blocked with PBS containing 1% BSA and anti-mouse CD16/32 (1:100, BioLegend) for 40 minutes at room temperature. Following the blocking step, the slides were incubated with a mixture of fluorochrome-conjugated antibodies for 2 hours at room temperature in a humid box. Finally, the slides were mounted using Prolong Gold containing DAPI (Life Technologies). The immunohistochemical analysis utilized the following anti-mouse antibodies: complement C3-FITC (1:200, Cedarlane, Cat# 1850362A); IgG2a-PE (1:200, BioLegend, Cat# 407107); CD3-APC (1:40, BioLegend, Cat# 100235); GL7-AF488 (1:80, BioLegend, Cat# 144606); and IgD-PE (1:100, BioLegend, Cat# 405705). Slides were examined and imaged using a Zeiss LSM880 confocal microscope. Image processing and quantification of the fluorescent intensity was performed with ImageJ and ZEN 2.1 Lite software equipped with a 20× objective.
5
Multiparametric Blood Cell Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Blood was collected from the inguinal veins at the indicated time points (Fig. 1A ) and hematological examination was conducted using an autohematology analyzer (Mindray BC-5000; Mindray, Shenzhen, China). Flow cytometric analysis was performed using a BD LSR Fortessa flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo v10.7.1 (BD Biosciences), as previously described (8 (link)). To exclude dead cells, blood was first stained with Fixable Viability Stain 575V (BD Biosciences) for 20 min at room temperature. For surface staining, cells were stained with the following antibodies for 30 min at 4°C: CD3 (Alexa Fluor 700; Invitrogen, Waltham, MA, USA), CD20 (APC/Cyanine7; Invitrogen), CD27 (PE/Cyanine7; Invitrogen), IgD (PE; BioLegend Inc., San Diego, CA, USA), IgM (FITC; SouthernBiotech, Birmingham, AL, USA), IgG (V450; BD Bioscience), CD4 (V500; Invitrogen), CD8 (V450; Invitrogen), CD95 (PE/Cyanine5; Invitrogen), and CD28 (ECD; Beckman Coulter, Brea, CA, USA). The cells were washed with permeabilization wash buffer and fixed with 1% paraformaldehyde. Data were acquired using an LSR Fortessa system (BD Biosciences) and analyzed using FlowJo v10.7.1 (BD Biosciences).
6
Immunohistochemical Analysis of Splenic and Renal Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Splenic and kidney sections were embedded in Tissue-Tek OCT compound (Sakura Finetek) and rapidly frozen in a freezing bath of dry ice and 2-methylbutane. Frozen OCT samples were cryosectioned and unstained slides were stored at −80°C. Immunohistochemical staining procedures were performed as previously described (1 (link), 2 (link), 5 (link)). For detection of germinal centers in the spleen, the following monoclonal Abs were used: GL7-AF488, IgD-PE, and CD4-allophycocyanin (all from BioLegend). For detection of renal deposition, C3-FITC (Cedarlane, Burlington, NC) and IgG2a-PE (eBioscience) Abs were used. Images were captured with a Zeiss LSM 880 confocal microscope (Fralin Imaging Center, Virginia Tech). Corrected total cell fluorescence scores were calculated with ImageJ software (National Institutes of Health, Rockville, MD).
7
Identifying Germinal Center Architecture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fixed single popliteal LNs were frozen in optimum cutting temperature compound and cut into 7-µm sections. Slides were blocked and incubated with fluorescently labeled antibodies. B220-PE, Streptavidin-647, IgD-PE, and GL-7-A647 antibodies were from BioLegend. CD35-biotin antibody was from BD Biosciences. GCs were identified as IgD− or IgD− GL-7+ areas. Where the number of available fluorescent channels exceeded the number of required labels, consecutive slides were stained with separate antibody panels. Images were captured using a 20× objective and a Ti2 eclipse inverted confocal microscope (Nikon). For imaging specifications, see smFISH: Hybridization and imaging. Quantification of cell number within GC DZ and LZ compartments was done using automatically detected nuclei/spots in Imaris software (v9.5.1, Oxford Instruments). Automatic threshold selection was manually verified, and non-B220+ cells were removed from analysis.
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!