Alexa fluor 488 conjugated goat anti rabbit igg

Detection of LDLR in HepG2 cells and liver tissues by immunofluorescence was performed as previously described [32 (link),41 (link)] with minor modification. Briefly, after treatment, HepG2 cells were rinsed with PBS for 5 min for 3 times and fixed in 4% (w/v) para-formaldehyde in PBS for 30 min. Liver tissues were fixed in 4% (w/v) para-formaldehyde in PBS at 4 °C for 48 h, embedded in paraffin and sliced at 4 μm thickness. After deparaffinization and hydration, tissue sections were pretreated by heating for 20 min in sodium citrate solution (0.01 M, pH 6.0) in a 95 °C water bath for the antigen retrieval. Thereafter, the cells or tissue sections were blocked with 10% (v/v) goat serum in PBST for 1 h and incubated with rabbit anti-LDLR antibody (1:100, Cat# ab52818) overnight at 4 °C, followed by incubation with Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1:200, Cat# D110061) for 1 h at room temperature and counterstained with DAPI (KeyGEN BioTECH, Nanjing, China) to show cell nucleus. Images were acquired by using Zeiss AX10 fluorescence microscopy (Zeiss, Oberkochen, Germany).

A cell climbing slice was prepared in a 24 well plate. When cells were attached to the wall, they were divided into a control group and salvianolic acid probe group. The cells were rinsed for 5 min with PBS buffer 3 times and then fixed in 3.7% formaldehyde at −4 °C for 10 min. The serum was diluted to 5% with room temperature PBS and incubated at room temperature for 30 min (closed liquid containing 0.1% Triton X-100). Primary antibodies used included rabbit anti-transgelin (dilution, 1:200; Abcam) and mouse anti-actin (dilution, 1:100; CST). After being rinsed gently with PBS three times, the cells were incubated at 37 °C for 1 h with Alexa Fluor® 488-conjugated goat anti-rabbit IgG (dilution, 1:150; KeyGEN BioTECH) and Alexa Fluor® 568-conjugated goat anti-mouse IgG (dilution, 1:250; Invitrogen) as secondary antibodies. The salvianolic acid probe was connected to 647 dyes by click reaction. The cells were viewed through a confocal microscopy system (Nikon, Japan).