Anti pna

For tissue samples, mesenteric lymph nodes (mLNs) were fixed with formalin and embedded with paraffin. The following antibodies were used for immunofluorescence analysis: anti-peanut agglutinin (anti-PNA, 20 μg/ml, Vector Laboratories), an anti-goat HRP-linked antibody (Abcam, UK), and an anti-CD3 antibody (Abcam, UK). Images were obtained using a laser scanning microscope (Olympus, Japan).

Testis fixed in 4% paraformaldehyde (PFA) at 4°C was dehydrated stepwise through an ethanol series and embedded in paraffin. About 5 μm sections were cut using Leica RM2235 Slicing Machine. After dewaxing and rehydration, sections were stained with H&E. For immunostaining, sections were boiled in 10 mM sodium citrate buffer (pH 6.0) for 20 min. On cooling to room temperature, samples were washed in PBS and blocked with 10% donkey serum in PBS for 1 h. Then sections were incubated with primary antibodies diluted in PBS containing 3% bovine serum albumin (BSA) at 4°C overnight and washed in PBS before incubating with secondary antibodies for 2 h at room temperature. After washing in PBS thoroughly, sections were incubated with DAPI and mounted on slides for immunofluorescence analysis in Leica DMR Fluorescence Microscope.
The antibodies used are as follows: anti-UCLH1 (Abcam, USA, Ms mAb; 1:400 dilution, 0.5 μg/ml), anti-SYCP1 (Santa Cruze, USA, Rb pAb; 1:100 dilution, 0.5 μg/ml), anti-SOX9 (Abcam, USA, Rb pAb; 1:400 dilution, 0.5 μg/ml), anti-PNA (Vector Labs, 1:500 dilution), Alexa-Fluor-488 Affinipure Goat Anti-Mouse IgG (Abcam, USA, 1:500, 4 μg/ml), Alexa-Fluor-555- Dnk pAb to Rb IgG (Abcam, USA, 1:500, 4 μg/ml).