Flow cytometry was used to analyze binding of rabbit antibodies to spleen leukocytes [9 (link)]. The cells in PBS containing 1% BSA were blocked with 10% (v/v) donkey serum (Southern Biotech, Birmingham, AL) before or after incubation with rabbit polyclonal anti-mutated peptide antibodies (1 × 107 cells/1.1 μg each Ab/100μL). Cell-bound rabbit Ig was detected with phycoerythrin-conjugated donkey anti-rabbit Ig (H + L) antibody (BioLegend, San Diego, CA). All the incubation steps were for 30 min in ice followed by three-time (3 min each) washings with PBS containing 1% BSA. The stained cells were fixed in 2% paraformaldehyde and following PBS washes, the samples were run on BD LSRII (BD Biosciences, San Jose, CA) flow cytometer equipped with BD FACSDiva™ version 8 software for data acquisition. The data were analyzed using FlowJo™ v10.1 software (FlowJo, LLC, Ashland, OR) following the required gating with the help of proper controls.
Donkey serum
Manufactured by Southern Biotech
Sourced in United States
Donkey serum is a biological fluid derived from the blood of donkeys. It contains a variety of proteins, growth factors, and other biomolecules that may be of interest for certain research and laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Facsdiva version 8, by BD (1 mentions)
Lsr 2, by BD (1 mentions)
Donkey serum, by GE Healthcare (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 596, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Southern Biotech (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
2 protocols using donkey serum
1
Flow Cytometry Analysis of Antibody Binding
2
Evaluating Graft Survival and Lesions
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Double/triple immunofluorescence labeling techniques were performed with direct-mounted sections to assess graft survival and lesion extent in vivo. Sections were washed in TBS, blocked in TBS/5% donkey serum (GE Healthcare)/0.25% Triton X-100 (Neolab) for 1 h and incubated with primary antibodies in TBS/1% donkey serum/0.25% Triton X-100 overnight at 4°C. The following day, sections were rinsed in TBS/1% donkey serum and incubated with Alexa Fluor 594 or Alexa Fluor 488 donkey secondary antibodies for 2 h. Sections were coverslipped with Fluoromount G (Southern Biotech, Birmingham, AL, USA).
The following primary antibodies were used: mouse anti-GFAP for astroglia (1:1,000; Merck Millipore), rabbit anti-GFP (green fluorescent protein, 1:750; Invitrogen) to detect the grafted cells, rabbit anti-Ki-67 (1:500; Novacastra/Leica Biosystems Wetzlar, Germany) for proliferating cells, and DAPI as nuclear counterstain. Immunolabeling was visualized using Alexa Fluor 488 (1:300; Life Technologies) or Alexa Fluor 596 (1:300; Life Technologies)-linked donkey secondary antibodies.
The following primary antibodies were used: mouse anti-GFAP for astroglia (1:1,000; Merck Millipore), rabbit anti-GFP (green fluorescent protein, 1:750; Invitrogen) to detect the grafted cells, rabbit anti-Ki-67 (1:500; Novacastra/Leica Biosystems Wetzlar, Germany) for proliferating cells, and DAPI as nuclear counterstain. Immunolabeling was visualized using Alexa Fluor 488 (1:300; Life Technologies) or Alexa Fluor 596 (1:300; Life Technologies)-linked donkey secondary antibodies.
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