Rabbit anti lrrk2

Proteins were loaded (50 μg), separated by 6, 8, and 15% SDS–PAGE and then transferred to polyvinylidene fluoride membranes (PVDF, Millipore, USA). The primary antibodies: rabbit anti-NLRC4 (1:800, Novus Biologicals, USA), mouse anti-pNLRC4 (1:400, ECM Biologicals, USA), mouse anti-RGS2 (1:500, Santa Cruz, USA), rabbit anti-Pro-caspase-1 (1:1000, Abconal, China), rabbit anti-Pro-IL-1β (1:1000, Abconal), rabbit anti-IL-18 (1:1000, Abconal, China), rabbit anti-cleaved caspase-1 (1:500, Cell Signaling, USA), rabbit anti-cleaved IL-1β (1:500, Affinity, USA), rabbit anti-cleaved IL-18 (1:200, R&D, USA), rabbit anti-TNF-α (1:500, Boster, China), rabbit anti-IL-6 (1:500, Boster, China), rabbit anti-LRRK2 (1:1000, Abcam, USA), rabbit anti-pLRRK2 s935 (1:500, Abcam, USA) and rabbit anti-β-actin (1:1000, Proteintech, USA). Secondary antibody (1:2000, Proteintech, USA)-linked horseradish peroxidase (HRP) was added to the bands for 2 h. ECL detection reagents (Thermo, USA) were used to visualize the bands. ImageJ software was used to determine the relative density of these proteins.

The primary antibodies used for immunocytochemistry were rabbit anti-OCT4 (1:500, Abcam), mouse anti-TRA-1-81 (1:250, BD Pharmingen), rabbit anti-TH (tyrosine hydroxylase, 1:250, Millipore), and mouse anti-TUBB3 (1:10,000, GeneTex). The secondary antibodies were Alexa594-conjugated donkey anti-rabbit IgG (1:200, Invitrogen) and Alexa488-conjugated donkey anti-mouse IgG (1:200, Invitrogen). The antibodies used for Western blotting were rabbit anti-α-synuclein (1:800, Proteintech), rabbit anti-phosphor-α-synuclein (Ser129) (1:1000, Abcam), rabbit anti-LRRK2 (1:1000, Abcam), rabbit anti-phospho-LRRK2 (Ser1292) (1:500, Abcam), and mouse anti-TUBB3 (1:10,000, Biolegend). The secondary antibodies were goat anti-mouse IgG-HRP (1:20,000, GE Healthcare) and goat anti-rabbit IgG-HRP (1:20,000, GE Healthcare).

Double immunofluorescence staining was conducted as described elsewhere (27 (link)). After washing thrice with 0.3% Triton in phosphate-buffered saline (PBS) to permeabilize the cell membrane, the frozen brain sections (15 μm thick) were blocked with an immunostaining blocking buffer (Beyotime) at room temperature for 1 h and then incubated at 4°C overnight using the following primary antibodies: rabbit anti-Rab10 (phospho T73) (1:200, Abcam), rabbit anti-LRRK2 (1:200, Abcam), and mouse anti-NeuN (1:300, Cell Signaling, USA). After incubating with Alexa Fluor 488 donkey anti-mouse IgG antibody (1:800, Invitrogen) and Alexa Fluor 555 donkey anti-rabbit IgG antibody (1:800, Invitrogen) at room temperature for 1 h, the sections were stained for 10 min with 4′, 6-dididia-2-phenylindolediamine hydrochloride (DAPI), and were assessed thereafter under a fluorescence microscope (OLYMPUS, U-RFL-T, Japan).