Fitc goat anti rabbit igg

(E)2’-hydroxychalcone (2’-HC; purity ≥ 98%) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Hydroxychloroquine (HCQ; purity ≥ 98%), 3-Methyladenine (3-MA; purity ≥ 98%), and N-acetylcysteine (NAC; purity ≥ 99%) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Rapamycin (Rap; purity ≥ 98%) was purchased from Aladdin (Shanghai, China).
The primary antibodies used were as follows: β-actin, Bax, Bcl-2, PARP, cleaved-PARP p25, caspase-9, caspase-3, LC3B, Beclin1, and p-IκB. They were purchased from ABclonal (Wuhan, China). p62/SQSTM1 was obtained from Proteintech (Wuhan, China). ERK, JNK, p-JNK, p38, p-p38, NF-κBp65, IκB, p-IκB, p-eIF2α, and MMP9 were obtained from Abmart (Shanghai, China). P-ERK, p-NF-κBp65, ATF-4, and CHOP were purchased from Wanleibio (Shenyang, China). HRP Goat Anti-Rabbit IgG (Abclonal), Alexa Flour 594-Goat Anti-Rabbit IgG (Abbox, Jiangsu, China), Cy3 Goat Anti-Rabbit IgG (H + L) (Abclonal), and FITC Goat Anti-Rabbit IgG (Servicebio, Wuhan, China) were used as secondary antibodies for a Western blot or immunofluorescence.

The tumor tissue specimens collected from DM and DM-4T1 groups were fixed in neutral-buffered 10% formalin. The samples were prepared for histological examinations. The CD8+ and CD4+ T-cell infiltration was investigated by immunofluorescence (IF). The primary anti-CD4 and anti-CD8 monoclonal antibodies and the secondary antibodies FITC-Goat Anti-Rabbit IgG and Cy3-AffiniPure™ Goat Anti-Mouse IgG were purchased from Servicebio (Wuhan, China). The DNA-specific dye DAPI (GDP1024, Servicebio, China) was used to label nuclear DNA. The slides were stained with hematoxylin and eosin (HE).

The sections from each group were used for immunofluorescence with GFAP and BrdU/DCX. For BrdU staining, sections were incubated with the following primary antibodies: mouse monoclonal anti-BrdU (1:1000, #5292, Cell Signaling Technology, USA) and rabbit polyclonal anti-DCX (1:2000, ab18723, Abcam, Cambridge, UK); polyclonal rabbit anti GFAP (1:200, #80788, Cell signaling technology, USA). Sections were then rinsed in PBS, followed by incubation with the following secondary antibodies: cyanine 3 goat anti rabbit IgG (GB21303, 1:500, Servicebio, Wuhan, China), cyanine 5 goat anti-rabbit IgG (GB27303, 1:500, Servicebio, China), and FITC goat anti-rabbit IgG (GB22303, 1:500, Servicebio, China) for one hour at room temperature. After washing, nuclei were counterstained with DAPI Fixed Cell Stain (Servicebio Co., Ltd., China). Sections were then mounted on slides and dehydrated. Finally, slides were coverslipped and kept in darkness at 40 °C for analysis.