Anti-SQSTM1 / p62 antibody (ab56416)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Three-μm thickness kidney cryosections were fixed for 15 min in 4% paraformaldehyde, followed by permeabilization with 0.2% Triton X-100 in 1× phosphate-buffered saline (PBS) for 5 min at room temperature. After blocking with 2% donkey serum for 60 min, the slides were immunostained with the primary antibodies. Cells cultured on coverslips were washed twice with cold 1× PBS and fixed with cold methanol/acetone (1:1) for 10 min at −20 °C. After three times of extensive washing with 1× PBS, the cells were treated with 0.1% Triton X-100 for 5 min, blocked with 2% normal donkey serum in 1× PBS buffer for 40 min at room temperature, and incubated with antibodies	Cells were also stained with 4′,6-diamidino-2-phenylindole to visualize the nuclei. Slides were viewed with a Nikon Eclipse 80i epi-fluorescence microscope equipped with a digital camera or Zeiss LSM710 (Zeiss).

Protocol tips
Three-μm thickness kidney cryosections were fixed for 15 min in 4% paraformaldehyde, followed by permeabilization with 0.2% Triton X-100 in 1× phosphate-buffered saline (PBS) for 5 min at room temperature. After blocking with 2% donkey serum for 60 min, the slides were immunostained with the primary antibodies. Cells cultured on coverslips were washed twice with cold 1× PBS and fixed with cold methanol/acetone (1:1) for 10 min at −20 °C. After three times of extensive washing with 1× PBS, the cells were treated with 0.1% Triton X-100 for 5 min, blocked with 2% normal donkey serum in 1× PBS buffer for 40 min at room temperature, and incubated with antibodies
Downstream tips
Cells were also stained with 4′,6-diamidino-2-phenylindole to visualize the nuclei. Slides were viewed with a Nikon Eclipse 80i epi-fluorescence microscope equipped with a digital camera or Zeiss LSM710 (Zeiss).

Publication protocol

Three-μm thickness kidney cryosections were fixed for 15 min in 4% paraformaldehyde, followed by permeabilization with 0.2% Triton X-100 in 1× phosphate-buffered saline (PBS) for 5 min at room temperature. After blocking with 2% donkey serum for 60 min, the slides were immunostained with the following: anti-FN (catalog no. F3648, Sigma); anti-α-SMA (catalog no. A5228, Sigma); anti-p-PKCα (Thr-638/641) (catalog no. 9375, Cell Signaling Technology); anti-LAMP2 (catalog no. L0668, Sigma); anti-SQSTM1/p62 (catalog no. ab56416, Abcam); anti-LC3B (catalog no. L7543, Sigma); and anti-laminin (catalog no. ab44941, Abcam). Cells cultured on coverslips were washed twice with cold 1× PBS and fixed with cold methanol/acetone (1:1) for 10 min at −20 °C. After three times of extensive washing with 1× PBS, the cells were treated with 0.1% Triton X-100 for 5 min, blocked with 2% normal donkey serum in 1× PBS buffer for 40 min at room temperature, and incubated with antibodies against p-PKCα, FN, α-SMA, LAMP2, SQSTM1/p62, or LC3 followed by staining with FITC or tetramethylrhodamine-conjugated secondary antibody. Cells were also stained with 4′,6-diamidino-2-phenylindole to visualize the nuclei. Slides were viewed with a Nikon Eclipse 80i epi-fluorescence microscope equipped with a digital camera or Zeiss LSM710 (Zeiss).

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