Anti rabbit secondary antibody

Multiplex immunofluorescence staining was also performed on 4-μm formalin-fixed paraffin-embedded sections. Antigen retrieval was performed with EDTA solution (pH = 9.0) at 95°C for 20 min after deparaffinizing the formalin-fixed paraffin-embedded sections. Human sections were blocked by 10% horse serum with 0.3 M glycine at room temperature for 1 h and then stained with primary antibodies targeting AR (1:200, rabbit, ab133273, abcam) at 4°C overnight. Secondary anti-rabbit antibody (Jackson ImmunoResearch Laboratories) was diluted at 1:2,000, and the sections were incubated at room temperature for 1 h. Horseradish peroxidase (HRP) was permanently fluorescence-labeled in secondary antibody by TYR-Cy3 (Recordbio) at room temperature for 10 min. Then, antigen retrieval was performed again with EDTA solution (pH = 9.0) at 95°C for 20 min. Human sections were blocked by 10% horse serum with 0.3 M glycine at room temperature for 1 h and then stained with primary antibodies targeting SYP (1:1,000, rabbit, ab32127, abcam) at 4°C overnight. Secondary anti-rabbit antibody (Jackson ImmunoResearch Laboratories) was diluted at 1:2,000, and the sections were incubated at room temperature for 1 h. HRP was permanently fluorescence-labeled in secondary antibody by TYR-488 (RecordBio) at room temperature for 10 min. DAPI (D9542, Sigma), diluted 1:1,000 in PBS, was used for nuclear staining.

Parental and csm2∆ cells were transformed with an empty plasmid or plasmid expressing either ALKBH2 or ALKBH2-CD. The cultures were grown in SC-URA medium overnight at 30°C. Subsequently, equal cell numbers (1 ml 0.75 OD₆₀₀) from each culture were pelleted, the supernatant was removed, and washed once with ddH₂O, and pelleted again. Protein was extracted from whole cell lysates by TCA preparation as described in 51 µl of loading buffer (Knop et al., 1999 (link)). The 13 µl of protein was run on a 10% SDS-PAGE gel and transferred to a PDVF membrane by semidry transfer (Bio-Rad) at 13 V for 90 min. ALKBH2 or Kar2 was Western blotted using αALKBH2 antibody (AbCam [ab154859]; 1:1000 with secondary antibody anti-rabbit [Jackson ImmunoResearch Laboratories 1:10,000]) or αKar2 antibody (Santa Cruz [sc-33630]; 1:5000 with secondary antibody anti-rabbit [Jackson ImmunoResearch Laboratories 1:10,000] as a loading control).

For immunofluorescence, RAW264.7 cells were seeded on cover slips and allowed to adhere overnight. After incubation with different intervention reagents for 24 h, the cells were washed three times with PBS and then fixed with 4% paraformaldehyde, permeabilized in 0.5% Triton-X100 for 30 min, and blocked with 1% BSA for 1 h. Cells were washed and incubated with anti-mouse iNOS (Abcam, ab15323), MR (Abcam, ab64693), VDR (Bioss, bs-2987R), and PPARγ (Bioss, bs-0530R) rabbit polyclonal antibodies overnight at 4°C. Then, cells were washed and incubated with anti-rabbit secondary antibody (Jackson, USA) for 2 h at room temperature. After staining nuclei with DAPI, cells were visualized using a IX70 fluorescence microscope (OLYMPUS, Tokyo, Japan).