Hrp goat anti rabbit igg

The antibodies against γH2AX (Cell Signaling Technology, Cat#80312S, Danvers, MA, USA), β-Tubulin (abcepta, Cat# AM1031A, San Diego, CA, USA), Caspase 9 (Proteintech, Cat#10380-1-AP, Rosemont, IL, USA), 53BP1 (Santa Cruz Biotechnology, Cat#SC-22760, Dalas, TX, USA), GAPDH (ABclonal, Cat#AC033, Woburn, MA, USA), Cyclin D1 (ABclonal, Cat#A19038), CDK2 (ABclonal, Cat#A0094), Flag (Proteintech, Cat#20543-1-AP, Rosemont, IL, USA), Top2α (ABclonal, Cat#A16440, Woburn, MA, USA), Top2β (Proteintech, Cat#20549-1-AP), HRP Goat Anti-Rabbit IgG (ABclonal, Cat#AS014, Woburn, MA, USA), HRP Goat Anti-Mouse IgG (ABclonal, Cat#AS003), Alexa Flour^TM 488 donkey anti-mouse IgG (H+L) (Thermo Fisher Scientific, Cat#A21202, Waltham, MA, USA), and Alexa Flour^TM 594 donkey anti-rabbit IgG (H+L) (Thermo Fisher Scientific, Cat#A21207) were purchased commercially.

(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.

All the tissue was fixed in 10% formaldehyde, dehydrated in ethanol, cleared in xylene, and embedded in paraffin. Sections (5 μm) were subjected to routine H&E stain and established the IHC staining technique using standard procedures. Rabbit mAb to IRF-1 (Cell Signaling Technology, #8474, USA), IRF-2 (Abcam, ab124744, USA), E-cadherin (ABclonal, A3044, China), N-cadherin (ABclonal, A0433, China), and Vimentin (ABclonal, A11423, China) were used as the primary antibodies and HRP Goat anti-rabbit IgG (ABclonal, China) was used as the secondary antibody.

The liver tissues from WT and fmo5^−/− loach were used for WB analysis. The polyclonal antibody of FMO5 and anti-GAPDH (Rabbit, AC001, ABclonal, China) were diluted 1000 and 2000 times with the primary antibody dilution buffer (Gbcbio Technologies Inc., China), respectively. The peroxidase-conjugated goat anti-rabbit IgG (HRP Goat Anti-Rabbit IgG, ABclonal AS014, 1:10000) was selected as the second antibody. Detailed procedures were described by Ida et al. [82 (link)].

L-theanine (CAS: 3081-61-6, 98% purity) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Donkey anti-Rabbit IgG H&L (Alexa Fluor 488) (#ab150075; 1:500) was obtained from Abcam (Cambridge, UK). DAPI was purchased from Invitrogen (Carlsbad, CA, USA). STO-609, BAPTA-AM were purchased from Topscience (Shanghai, China).
Primary antibodies against ACC1 (#3676), FASN (#3180), p-AMPK (#2535), AMPKα (#5831) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody against p-CaMKKβ (#AF4487), CaMKKβ (#DF4793) were purchased from Affinity Biosciences (OH, USA). Antibody against SREBP-1c (#A15586), phospho-mTOR (#AP0094), mTOR (#A2445), HRP Goat Anti-Rabbit IgG (#AS014), and HRP Goat Anti-Mouse IgG (#AS003) were purchased from ABclonal (Wuhan, Hubei, China). Antibodies against PPARα (#15540-1-AP), CPT1A (#15184-1-AP), SREBP-1c (#14088-1-AP) were purchased from Proteintech (Chicago, IL, USA). Antibodies recognizing GAPDH (#AP0063), Lamin B (#AP6001), and β-ACTIN (#AP0060;) were purchased from Bioworld Technology (Minneapolis, MN, USA). Donkey anti-Rabbit IgG H&L (Alexa Fluor 488) (#ab150075) was obtained from Abcam (Cambridge, UK). DAPI was purchased from Invitrogen (Carlsbad, CA, USA).

The paraffin sections of gastric tissue were treated with 3% hydrogen peroxide at room temperature for 15 min and washed 3 times with PBS, 5 min each time. Following goat serum blocking at room temperature for 30 min, the sections were taken out, dried the sealing solution, and incubated with primary antibodies at 4°C overnight. The primary antibodies were as follows: anti-Raf-2 (122361 000, Abcam), anti-MEK1 (1:1 000, Abcam), anti-MEK2 (1:1 000, Abcam), and anti-ERK1 (1: 1 000, Abcam). The specimens were washed with PBS 3 times, 5 min each time. Secondary antibodies (HRP Goat Anti-Rabbit IgG, 1:1 000, ABclonal) were incubated at room temperature for 1.5 h. The specimens were washed with PBS 3 times, 5 min each time. After DAB development and hematoxylin re-staining, the staining was observed under a microscope.

The following antibodies were used: anti-Bcl-2 pAb (Bioss, Proteintech Group), anti-Cytochrome C Rabbit pAb (ABclonal Technology), anti-Bax Rabbit pAb (Wanlei Biotechnology Co., Ltd), anti-Bak Rabbit pAb (ABclonal Technology), anti-caspase-3 Rabbit pAb (ABclonal Technology), anti-caspase7/cleaved-caspase7 Rabbit pAb (ABclonal Technology), IL-1β (Wanleibio Biotechnology Co., Ltd), anti-aggrecan Rabbit pAb (ABclonal Technology), Coll1A2 Rabbit pAb (ABclonal Technology), anti-GAPDH Rabbit pAb (ABclonal Technology), HRP goat anti-rabbit IgG (ABclonal Technology), Alexa Fluor® 488 labeled goat anti-rabbit IgG (Servicebio Technology Co., Ltd), Cy3 labeled goat anti-rabbit IgG (Servicebio Technology Co., Ltd).