Alexa fluor 594 goat anti rabbit igg

The rabbit anti-UL21 polyclonal antibodies were generated for this study, and the rat anti-UL16 polyclonal antibodies were provided by He Qin [20 (link)]. The following monoclonal antibodies were used in this study: rabbit anti-GRP78 BiP (Abcam, UK), mouse anti-TGN46 (Abcam, UK), goat anti-rabbit IgG (Thermo Fisher Scientific, USA), rabbit anti-Myc tag (Beyotime, CHN), mouse anti-Flag tag (Transgen Biotech, CHN), Alexa Fluor 594 goat anti-rabbit IgG (Thermo Fisher Scientific, USA), Alexa Fluor 488 goat anti-mouse IgG (Thermo Fisher Scientific, USA), Alexa Fluor 488 goat anti-rat IgG (Abcam, UK), Alexa Fluor 594 goat anti-rabbit IgG (Life Technologies, USA), and mouse anti-β-actin (Beyotime, CHN). Normal rabbit IgG was obtained from Beyotime, and normal rat IgG was obtained from Thermo. The pCAGGS [40 (link)], pCMV-Myc [41 (link)], and pET-32c plasmids were provided by the Sichuan Agricultural University Avian Diseases Research Center.

EWAT, liver, pancreas, and intestinal were harvested and fixed overnight in 4% paraformaldehyde, paraffin-embedded, then sectioned (5 μm), followed by hematoxylin and eosin (H&E) staining. Adipose tissue sections were hybridized with CD3 (Cat#AF20162, AiFang, Changsha, China) and F4/80 (Cat#SAF002, AiFang, Changsha, China) antibodies. Alexa Flour 488 donkey anti-mouse IgG (Cat# A32766, Invitrogen, USA) was used as a secondary antibody to detect CD3⁺ cells. Alexa Fluor 594 goat anti-rabbit IgG (Cat#A32740, Invitrogen, USA) was used as a secondary antibody to detect F4/80⁺ cells. For the detection of macrophage apoptosis, the TUNEL assay was performed using a FITC TUNEL cell apoptosis detection kit (Cat# G1501-100T, Servicebio, Wuhan, China).
Pancreatic paraffin-embedded tissue sections were stained with mouse anti-insulin (Cat# 66198-1, Proteintech, USA), rabbit anti-Glucagon (Cat#ab92517, Abcam, The UK), rabbit anti-Ki67 (Cat# D3B5, Cell Signaling Technology, USA) antibodies. Alexa Flour 488 donkey anti-mouse IgG (Cat# A32766, Invitrogen, USA) and Alexa Fluor 594 goat anti-rabbit IgG (Cat# A32740, Invitrogen, USA) were used as secondary antibodies. Intestinal paraffin-embedded tissue sections were stained with mouse anti-GLP1 (Cat# sc-514592, Santa Cruz, USA). Images were acquired with an Olympus microscope and integrated density was analyzed with Image J Software.

The paraffin sections were dewaxed using xylene for 15 min three times and hydrated in 100%, 90%, 80%, and 70% EtOH. Antigen retrieval was performed by a microwave in boiling antigen retrieval solution (pH 6.0, Dako, CA, USA) for 2 min 20 s. The sections were stained with the rabbit Ki67 antibody (Abcam, IA, USA, 1:300) overnight at 4 °C, and then incubated with secondary antibodies, such as Alexa Fluor 594 goat anti-rabbit IgG or Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen, NY, USA, 1:1000), for 1 h at room temperature with 4,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). For cell staining with phospho-EGFR1, phospho-ErBb4, or phospho-Hck antibodies, the cells were fixed with 4% paraformaldehyde for 30 min at room temperature; washed with PBS; and incubated with phospho-EGFR1 (Abcam, 1:300), phospho-ErBb4 (Abcam, 1:300), or phospho-Hck (Abcam, 1:300) antibodies overnight at 4 °C. The samples were then incubated with Alexa Fluor 594 goat anti-rabbit IgG or Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen, NY, USA, 1:1000) secondary antibodies for 1 h at room temperature with DAPI. Images of immunofluorescence staining were captured using a ZEISS LSM700 confocal microscope.