Envision hrp secondary antibody

Four-μm-thick sections were deparaffinized, rehydrated in serially graded ethanol, heated in citric buffer (pH 6.0) once for 20 min in a microwave oven for antigen retrieval, and blocked with 3% hydrogen peroxide for 15 min. The samples were then labeled with anti-TEM8 antibody (1:300, Abcam, #21270), anti-cyclin D1 antibody (1:200, cell signaling technology) and anti-pERK1/2 antibody (1:200, cell signaling technology) at 4 °C overnight. The next day, after washing with phosphate-buffered saline (PBS), the sections were incubated with EnVision-HRP secondary antibody (DAKO, Carpinteria, CA) for 30 min at 37 °C in a water bath, washed with PBS, stained with 0.5% diaminobenzidine and counterstained with Mayer’s hematoxylin, then air dried, and mounted with resinene.

Paraffin-embedded samples of human fat tissue were selected for immunohistochemical and immunofluorescence analysis. Samples were deparaffinized with xylene and rehydrated by an increasing ethanol gradient for hematoxylin and eosin (H&E) staining. Target retrieval was performed using the PT Link (DAKO) at pH solution 9.0 (DAKO). Immunohistochemistry staining was performed using a Dako Autostainer Link 48. Primary antibodies used were as follows: rabbit monoclonal ALP (Abcam, 1/200), mouse monoclonal CD73 (Abcam, 1/200), mouse monoclonal CD31 (DAKO, 1/200), and the appropriate EnVision HRP secondary antibody (EnVision HRP rabbit or mouse, DAKO, 1/500) according to the manufacturer’s instruction. Immunofluorescence was performed using a Dako Autostainer Link 48 with the following antibodies: rabbit monoclonal ALP (Abcam, 1/200), mouse monoclonal CD73 (Abcam, 1/200), Alexa Fluor 488 goat anti-rabbit IgG (Thermo Fisher, 1/200), and Alexa Fluor 546 goat anti-mouse IgG (Thermo Fisher, 1/200) according to the manufacturer’s instruction. Sections were visualized with LEICA DM6600 with a × 20 magnifying objective lens.

Standard immunohistochemistry procedures were conducted on 5 μm tissue sections prepared from formalin-fixed paraffin-embedded tissue blocks. Briefly, the sections were deparaffinized and rehydrated in xylene and alcohol, followed by antigen retrieval in 0.01 M sodium citrate buffer (pH 6.0) heated in a microwave oven. Afterward, the tissue sections were incubated with an anti-EFEMP1 antibody (1 : 50 dilution, AP9095a, Abgent, San Diego, CA) in a humidified container at 4°C overnight. The negative control sections were incubated with PBS. Thereafter, the tissue sections were incubated with EnVision-HRP secondary antibody (Dako, Carpinteria, CA). At least five randomly selected regions from each tissue section were scored. The integral optical density and area were quantified using Image-Pro Plus 6.0 software. The average of five optical density values was used to represent the expression intensity for each section.