The number of IgM-secreting cells was quantified by ELISPOT. Multiscreen 96-well filter plates (Millipore, Billerica, Massachusetts) were coated with anti-human IgM antibody (5 μg/ml) (Sigma Aldrich, St. Louis, MO) overnight and subsequently blocked with 5% bovine serum albumin (Sigma Aldrich, St. Louis, MO) for 2 h. Cells were washed with RPMI 1640 twice, resuspended in RPMI 1640 containing 10% bovine calf serum (Thermo Scientific, Lafayette, Colorado) and incubated on the primary antibody-coated plates overnight at 37°C with 5% CO2. Biotin-conjugated anti-human IgM antibody (Sigma Aldrich, St. Louis, MO) and streptavidin horseradish peroxidase (HRP) (Sigma Aldrich, St. Louis, MO) were added for one-hour and incubated at 37°C with 5% CO2. All incubations were followed by three washes with phosphate-buffered saline (pH 7.4) containing 0.1% Tween-20 (Sigma Aldrich, St. Louis, MO) and three washes with nanopure water. The spots were developed with an aminoethylcarbazole staining kit (Sigma Aldrich, St. Louis, MO). The number of spots per well between 0.0001mm2 and 9.6372mm2 were quantified via the Immunospot Software (Cellular Technology, Ltd, Shaker Heights, Ohio) and normalized to the number of viable cells collected from each well.
Aminoethyl carbazole staining kit
Manufactured by Merck Group
The Aminoethyl carbazole staining kit is a laboratory product designed for the detection of peroxidase activity in biological samples. The kit contains the necessary reagents and instructions for performing the staining procedure.
Automatically generated - may contain errors
Lab products found in correlation
Bovine calf serum (bcs), by Thermo Fisher Scientific (3 mentions)
Multiscreen 96 well filter plate, by Merck Group (3 mentions)
Immunospot software, by Cellular Technology (3 mentions)
Tween 20, by Merck Group (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Streptavidin horseradish peroxidase, by Merck Group (2 mentions)
Mouse anti human igm antibody, by Merck Group (2 mentions)
Biotin conjugated mouse anti igm, by Merck Group (2 mentions)
Streptavidin horseradish peroxidase hrp, by Merck Group (1 mentions)
Anti human igm antibody, by Merck Group (1 mentions)
Phosphate buffered saline ph 7.4, by Merck Group (1 mentions)
Extravidin horseradish peroxidase, by Merck Group (1 mentions)
Biotinylated goat anti mouse igg, by Novus Biologicals (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
4 protocols using aminoethyl carbazole staining kit
1
Quantification of IgM-Secreting Cells by ELISPOT
2
Quantification of IgM-secreting B Cells
3
Quantifying Neovascularization via CD31 Immunostaining
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Sections were immunostained for CD31, to indicate neovascularization, by incubating them with primary mouse anti-CD31/PECAM-1 monoclonal antibody (dilution, 1:150; cat. no. NB100-1642, Novus Biologicals, LLC, Littleton, CO, USA) and biotinylated goat anti-mouse IgG (dilution, 1:150; cat. no. NBP1-97590; Novus Biologicals, LLC). The sections were then incubated with ExtrAvidin-horseradish peroxidase (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Aminoethylcarbazole was used as a chromogenic substrate using an Aminoethylcarbazole Staining kit (Sigma-Aldrich; Merck KGaA). Microphotographs were captured, and 20 random fields of 3 stained sections (>4 fields/section) from each group were observed at ×40 magnification from central healing areas for semiquantitative analysis of microvessel density. Negative control sections were incubated with PBS instead of the primary antibody.
4
Quantifying IgM-Secreting B Cells
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In brief, multiscreen 96-well filter plates (Milipore, Burlington, MA) were coated with 5 μg/mL of purified mouse anti-human IgM antibody (Sigma-Aldrich) overnight, washed and blocked with 5% bovine serum albumin (BSA) (Sigma Aldrich) for 2 hours. During this time, B cells were harvested and washed 3x with RPMI 1640, enumerated, and resuspended in supplemented RPMI with 10% bovine calf serum (ThermoFisher) and incubated overnight at 37°C. The following day, filter plates were incubated with biotin-conjugated mouse anti-IgM (Sigma Aldrich) and streptavidin-horseradish peroxidase (Sigma Aldrich) for 1 h at 37°C. All incubations were preceded by 3 washes with phosphate buffered saline plus 0.1% Tween-20 (Sigma Aldrich) and 3 washes with nanopure water. IgM positive spots were developed with aminoethyl carbazole staining kit (Sigma Aldrich). Spots were quantified using Immunospot Software (Cellular Technology, Shaker Heights, OH) and normalized to the number of viable cells plated in each well.
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