Spleen segments were embedded in O.C.T. compound (Sakura), cut in 12µm thick sections and mounted on polysine coated slides (Thermo Scientific). Sections were fixed in ice-cold 4% PFA for 20 minutes at 4°C, washed, then permeabilized in ice-cold acetone for 10 minutes. After air drying, samples were rehydrated in PBS, blocked with 2% normal mouse and rat serum in staining buffer (PBS with 1% BSA) then stained with anti-CD4 (BD 560468), anti-Foxp3 (eBioscience 13-5773-80) and anti-IgD (eBioscience 17-5993) in staining buffer overnight at 4°C in a humid chamber. Sections were washed and biotin was detected with streptavidin-AF546 (Molecular Probes S-11225). After a final wash, sections were mounted with a coverslip and Vectashield antifade mounting medium (Vector Laboratories), then stored at 4°C in the dark until required for imaging.
S 11225
Manufactured by Thermo Fisher Scientific
The S-11225 is a laboratory equipment product offered by Thermo Fisher Scientific. It is designed to perform a specific core function within a laboratory setting. The concise and factual details about this product are limited in the information provided, and an unbiased description cannot be provided without the risk of extrapolation or interpretation.
Automatically generated - may contain errors
Lab products found in correlation
Ez link sulfo nhs biotin, by Thermo Fisher Scientific (2 mentions)
Polysine coated slides, by Thermo Fisher Scientific (1 mentions)
Anti foxp3, by Thermo Fisher Scientific (1 mentions)
Vectashield antifade mounting medium, by Vector Laboratories (1 mentions)
L1516, by Merck Group (1 mentions)
Fluoromount medium, by Southern Biotech (1 mentions)
Eclipse 90i microscope, by Nikon (1 mentions)
4 protocols using s 11225
1
Immunofluorescent Staining of Spleen Tissue
2
Immunolabeling of Otx2 and WFA
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For immunostaining, 50 µm floating sections were incubated with anti-Otx2 (rat polyclonal, 1/200, in-house) and biotinylated-WFA (1/100, Sigma L1516) in TBS, 1% Triton-X, 0.2% Tween-20, 10% fetal calf serum, overnight at 4°C. Sections were extensively washed at RT, incubated 2 h at RT with anti-rat Alexa Fluor-488 (Molecular Probes A21208, 1/2,000) and streptavidin Alexa Fluor-546 (Molecular Probes S11225, 1/2,000), washed again and finally mounted in Fluoromount medium (SouthernBiotech). Images were acquired with an Eclipse 90i microscope (Nikon).
3
Intestinal Epithelial Barrier Integrity Assessment
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Biotin was used as a tracer molecule to determine the integrity of the epithelial barrier20 (link). Briefly, immediately after removal of the entire intestinal tract from mock, C227 or Δstx2 infected rabbits, the colonic tissue was separated while maintaining their correct orientation (ie proximal vs distal ends). EZ-Link Sulfo-NHS-Biotin (Thermo Scientific, IL) was injected slowly (1–2 min) into the lumen via the open (cut) end of the most distal part of the colon. After 3 min, the tissue section just proximal to the site of injection was removed, fixed in 4% paraformaldehyde and processed for immunofluorescence staining as described above. Tissue sections were incubated with streptavidin linked to Alexa 546 (1/500) (S11225; Invitrogen) for 1 hr at room temperature before being counterstained with phalloidin Alexa fluor 633 and DAPI. Colonic tissue from mock, C227 or Δstx2 infected rabbits that were not treated with biotin exhibited no background staining following incubation with streptavidin only.
4
Intestinal Epithelial Barrier Integrity Assessment
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