Anti vimentin antibody

For Mafb expression analysis, adult Mafb-GFP knock-in mice in which the GFP gene was inserted into one allele of the Mafb locus [20 (link)] were used for the flow cytometric analysis as previously described [30 (link)]. Briefly, testes were treated twice with 1 mg/ml collagenase in DMEM containing 25 U/ml DNaseI twice at room temperature for 10 min and then with 2 mg/ml hyaluronidase and 1 mg/ml collagenase in DMEM containing 25 U/ml DNaseI at 32°C for 30 min with agitation. After filtration using a 35-μm filter, cells were suspended in PBS containing 2% fetal bovine serum (FBS) and 100 μg/ml propidium iodide (PI). PI-negative cells were sorted against GFP with a Beckman Coulter Gallios (Beckman Coulter), and Sertoli cells were distinguished from germ cells according to the parameters explained previously [30 (link)]. WT mice were used as a control. The data were analyzed using FlowJo software (Tree Star, Inc).
For RNA-Seq analysis, Sertoli cells from Mafb-cKO mice were sorted by the same method. Mafb^fl/fl mice were used as a control. The purity of the isolated cells was confirmed by immunocytochemical staining with anti-vimentin antibody (Progen Biotechnik; GP53).

Isolated Sertoli cells were plated onto a silanized glass slide using a Shandon Cytospin^™ 3 centrifuge. Cells were then fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. Cells were washed with PBS and blocked with 3% skim milk plus 0.1% Triton X-100 in PBS. Cells were then incubated with a 1:500 dilution of guinea pig polyclonal anti-vimentin antibody (Progen Biotechnik; GP53) overnight at 4°C. After washing, cells were incubated with a 1:1000 dilution of Alexa Fluor 594-conjugated secondary antibody (Life Technologies, Gaithersburg, MD, USA) for an hour at room temperature. Cells were counterstained with DAPI.