Purified Rat Anti-Mouse CD24 Clone M1/69 (RUO)

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Adult WT mouse colonic tissue was dissected and flushed with Mg2+/Ca2+-free HBSS. Segments (4 mm) of midproximal and middistal colon were fixed in 4% PFA followed by 30% sucrose and embedded in Tissue-Tek optimal cutting temperature (OCT) embedding medium for frozen tissue specimens (Sakura Finetek). Sections (7 μm) were incubated with Protein Block solution (Dako) for 1 h and then with purified rat anti-mouse CD24 primary antibody (1:50 dilution; catalog no. 557436, BD Bioscience) at 4°C overnight. After they were washed, the samples were blocked with 3% peroxide in methanol at room temperature for 10 min, washed, and incubated in biotinylated mouse anti-rat IgG2b,κ secondary antibody (1:200 dilution; catalog no. 553987, BD Bioscience) at room temperature in a humidity chamber for 1 h. Then the samples were washed and incubated in avidin-biotin complex (1:50 dilution; Vectastain Elite ABC Kit, Vector Laboratories) at room temperature in a humidity chamber for 30 min. The samples were washed again, and diaminobenzidine (Zymed Kit, Invitrogen) was applied to the slides. After completion of the reaction, the slides were placed in distilled water for 4 min and then counterstained with hematoxylin. Slides were then dehydrated, coverslips were applied using DPX mountant (Crown Scientific), and the slides were allowed to dry overnight and analyzed by light microscopy. For quantification of CD24 expression as a proportion of crypt length, 20 well-oriented crypts per animal (n = 5) were measured for total crypt length and length of CD24 expression.

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