Alexa fluor 594 conjugated goat anti rabbit igg secondary antibody

Alveolar macrophages were identified by CD68 surface markers, and M1 or M2 macrophages were distinguished using iNOS and arginase-1 surface markers, respectively. Paraffin-embedded lung tissue sections (4 μm) of the TB patients were pretreated with ethylenediaminetetraacetic acid (EDTA) antigen-retrieval citrate buffer (C9999; Sigma Aldrich) at 120 °C for 4 min in a pressure boiler. The sections were incubated with anti-CD68 (sc-20,060, 1:100; Santa Cruz Biotechnology), anti-iNOS (sc-651, 1:100; Santa Cruz Biotechnology) and anti-arginase-1 (sc-20,150, 1:100; Santa Cruz Biotechnology) primary antibodies overnight at 4 °C. Immunoreactivity was detected using Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody (A-11001, 1:200; Thermo) and Alexa Fluor 594-conjugated goat anti-rabbit IgG secondary antibody (A-11012, 1:200; Thermo). The slides were then mounted.

CD68 (28058-1, Proteintech, Wuhan, China) expression in arterial lesions was examined by immunofluorescence using 7-mm cryosections of the aortic root. The sections were blocked with 5% bovine serum albumin (BSA) for 1 h and incubated overnight at 4 °C with CD68 antibody (1:100). After washing, the sections were incubated with the Alexa Fluor 594-conjugated goat anti-rabbit IgG secondary antibody (1:200) (A11032, ThermoFisher, Waltham, MA, USA) for 1 h. Nuclei were counterstained with DAPI (1:1000) (C1002, Beyotime, Shanghai, China) for 5 min. The fluorescence signal was monitored by confocal laser scanning microscopy (Zeiss Microsystems, Jena, Germany).

To estimate oxidative injury after hypoglycemia, we conducted immunofluorescence staining with 4-hydroxy-2-nonenal (4HNE) antibodies (diluted 1:500, Alpha Diagnostic Intl. Inc., San Antonio, TX, USA) as described previously [4 (link)]. Brain sections were incubated in a mixture of polyclonal rabbit anti-HNE anti-serum and phosphate buffered saline (PBS) containing 0.3% TritonX-100 overnight in a 4 °C incubator. After washing three times for 10 min each with PBS, samples were also immersed in a mixture of Alexa Fluor 594-conjugated goat-anti rabbit IgG secondary antibody (diluted 1:250, Invitrogen, Grand Island, NY, USA) for two hours at room temperature. Finally, the sections were placed on gelatin-coated slides. To measure the oxidative stress signals, we used ImageJ using the following order of commands: click the menu option Image → Adjust → Color threshold and dark background. Then, we modulated brightness according to the intensity of oxidative stress. The image was converted to 8-bit images (magnification = 10×). The fluorescence intensity was expressed as mean gray value.

To evaluate level of oxidative stress in the hippocampus, we conducted immunofluorescence staining with paraformaldehyde-fixed brain tissue. Oxidative stress was detected by measuring the presence of the lipid peroxidation product, 4HNE (4-hydroxy-2-nonenal). Immunohistochemistry with 4HNE (Alpha Diagnostic Intl. Inc., San Antonio, TX, USA) antibodies was conducted according as previously described manual [64 (link)]. Brain sections were immersed in a polyclonal rabbit anti-4HNE serum (diluted 1:500, Alpha Diagnostic Intl. Inc., San Antonio, TX, USA) with the PBS containing 0.3% TritonX-100 for overnight in a 4 °C incubator. After we washed the sections three times for 10 min with PBS, these sections were also immersed in a solution of Alexa Fluor 594-conjugated goat anti-rabbit IgG secondary antibody (diluted 1:250, Invitrogen, Grand Island, NY, USA) for two hours at RT. The sections were laid on gelatin-coated slides in order to observe under a microscope. To measure the oxidative injury, we used Image J (v. 1.47c.) program and measured the mean gray value [63 (link)].

Formononetin was obtained from Yick-Vic Chemicals & Pharmaceuticals Ltd, (Hong Kong). Antibodies for GSK-3β and adenine nucleotide translocase (ANT) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against phospho-GSK-3β (Ser9), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), Akt, and phospho-Akt were obtained from Cell Signaling Technology (Boston, MA, USA). Anticyclophilin D (anti-Cyp-D) was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Alexa Fluor 594-conjugated goat anti-rabbit IgG secondary antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody were obtained from Invitrogen (Carlsbad, CA, USA).