Rabbit anti mkp 1

The cells were lysed with a protein lysis buffer (Pro-PREP, Intron Biotechnology, Korea) with a protease/phosphatase inhibitor cocktail, and protein concentration were determined using a DC assay kit (Bio-Rad, Berkeley, USA). The proteins were separated via 10-15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes, blocked with 3% bovine serum albumin in Tris-buffered saline with Tween (TBST: 20 mM Tris-HCl [pH 7.6], 137 mM NaCl, 1% Tween 20), and probed with the indicated primary antibodies; rabbit anti-p16^INK4a (1:1000; Abcam, Cambridge, UK; ab108349), rabbit anti-BMI1 (1:1000, Cell Signaling Technology Europe, Leiden, The Netherlands; #6964s), mouse anti-GAPDH (1:3000, Millipore, Darmstadt, Germany; MAB374), rabbit anti-COX-2 (1:1000, Abcam; ab15191), rabbit anti-p-p38 (1:1000, Cell Signaling; #9211s), rabbit anti-MKP-1 (1:200, Santacruz, Texas, USA; sc-1102). The secondary antibodies were used according to the manufacturer's specifications; horseradish peroxidase (HRP)-conjugated antibodies (1:2000; Invitrogen, Carlsbad, USA; G21040, G21234) and binding was detected using an enhanced chemiluminescence (ECL) detection kit (Amersham Pharmacia Biotek, Amersham, UK).

Mice at 7, 14, 21, 30 and 60 days pp were sacrificed by cervical dislocation, and testes were immediately removed and fixed with Bouin's solutions at room temperature overnight (day 0 was birth date). We used 4 mice per group. Then, 5 μm-thick serial paraffin sections were prepared. A number of paraffin sections were stained with haematoxylin staining using standard protocols, and other sections were immunostained using the following procedure. For antigen retrieval, the sections were incubated in buffered citrate (pH 6.0) for 15 minutes at 105°C for MKP-1. The samples were then soaked in methanol containing 0.3% H₂O₂ to neutralize endogenous peroxidase activity. Sections blocked in 10% normal rabbit serum for 30 minutes at room temperature were incubated with rabbit anti-MKP-1 (1:200; Santa Cruz Biotechnology) at 4°C overnight. On the following day, the sections were incubated with a polyclonal goat anti-rabbit IgG antibody. The sections were visualized using a Histostain-Plus Kit (LAB-SA Detection; System Invitrogen Life Technologies) according to the manufacturer's instructions for the secondary antibody and subsequent steps. Testis seminiferous epithelium stage analysis was performed as previously described [47 (link)].