Donkey anti goat igg h l

Equal amounts of protein (30 μg per lane) were subjected to 4–20% SDS-PAGE and then transferred to PVDF membranes. After blocking with 5% non-fat dry milk for 1.5 hours at room temperature, the membranes were incubated at 4 °C overnight with the following primary antibodies: pyruvate dehydrogenase kinase-4 (PDK4; 1:1000; NOVUS Biologicals) and β-actin (1:4000; Santa Cruz Biotechnology). The membranes were washed thrice for 10 min each with PBST at room temperature. Subsequently, the membranes were incubated with goat anti-rabbit IgG H&L (HRP; 1:1000; Abcam), donkey anti-goat IgG H&L (HRP; 1:1000; Abcam), or goat anti-mouse IgG H&L (HRP; 1:1000; Abcam) secondary antibodies for 2 hours at room temperature, and signals were visualized using an ECL detection system (Amersham Pharmacia Biotech, Inc.). The results were quantified by densitometric analysis using Image J.

Calcium silicide (CaSi₂, 95%, Gelest), iodine (I₂, 99.99%, Sigma‐Aldrich), acetonitrile (CH₃CN, 99.8%, Sigma‐Aldrich), 1‐methyl‐2‐pyrrolidinone (NMP, 98%, Sigma‐Aldrich), double distilled water (Milli‐Q system, Millipore, USA), hexadecyltrimethylammonium chloride (CTAC, 99%, Adamas), triethylamine (TEA, 99%, Adamas), tetraethyl orthosilicate (TEOS, SiO₂ iOrolidinGreagent), ethanol absolute (C₂H₆O, ≥ 99.7%, Shanghai Lingfeng Chemical Reagent Co. Ltd), hydrochloric acid (HCl, 36.0–38.0%, Shanghai Lingfeng Chemical Reagent Co. Ltd), and RP hydrochloride monohydrate (Adamas, 99%) were used. Antibodies which were used included Iba‐1 (WAKO, 019–19741), GFAP (Abcam, ab53554), TRPV1 (Abcam, ab5566), c‐Fos (CST, 2250S), Donkey Anti‐Rabbit IgG H&L (Abcam, ab150073), and Donkey Anti‐Goat IgG H&L (Abcam, ab150132). All chemicals used in slice preparation and electrophysiological recording were purchased from Sigma‐Aldrich.

The hypothalamic ARC tissues of rats in each group were homogenized using frozen RIPA lysis buffer and protease inhibitors. After ensuring complete tissue lysis, the solution containing total proteins was obtained by centrifugation, and the protein concentration was determined using the BCA protein detection kit (Thermo Fisher, USA). Electrophoresis was performed, and the separated samples were transferred via a membrane. To this membrane, the primary antibody solutions of Kiss1 (1:1000, Thermo Fisher, USA), Kiss1r (1:1000, Thermo Fisher, USA), AR (1:1000, Novus, USA), and ERα (1:1000, Novus, USA) were added and incubated overnight at 4 °C. Then, the secondary antibody donkey anti-goat IgG-H&L (1:3000, Abcam, UK) was added, and the protein antibody complexes were visualized using the ECL Chemiluminescence Visualization Kit (Servicebio, Wuhan, China). After the exposed film was developed and fixed, it was arranged using PhotoShop. The Alpha software (Innotech alphaEaseFC, USA) processing system was used to analyze the gray value of the target protein. The relative expression of Kiss1, Kiss1r, AR, and ERα was calculated by comparing the gray value ratio to the internal reference glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

All tissue sections were deparaffinized in xylol, dehydrated in graded ethanol dilutions, and washed three times in desilted water. The sections were heated in citrate buffer for antigen retrieval for 15 min. After cooling, tissue sections were incubated with the combination of primary antibodies RIP5 (N-16) (diluted 1:50, Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc 162109) and VGLL4 (diluted 1:50, Invitrogen, Waltham, MA, USA PA5-58467) overnight at +4 °C. Primary antibodies were washed in PBS three times and incubated with a combination of secondary antibodies: Goat Anti-Rabbit IgG H&L (Abcam, Cambridge, UK, Alexa Fluor^® 594) (ab150080) and Donkey Anti-Goat IgG H&L (Abcam, Cambridge, UK, Alexa Fluor^® 488) (ab150129) in Dako Antibody Diluent (Dako, Glostrup, Denmark, S0809) for 1 h at room temperature. After incubation with secondary antibodies, sections were washed in PBS three times, counterstained with DAPI, and coverslipped (Immuno-mount, Shandon Inc., Pittsburgh, PA, USA).

The IHC antibody anti-TFEB (ab2636) was purchased from Abcam. Western-blot antibodies, including anti-LAMP1 (sc-20011, Santa Cruz Biotechnology), anti-LC3 (3,868, Cell Signaling Technology), anti-p-P65 (3,033, Cell Signaling Technology), anti-P65 (8,242, Cell Signaling Technology), anti-p-S6 (4,858, Cell Signaling Technology), anti-S6 (2,217, Cell Signaling Technology), anti-p-4EBP1 (2,855, Cell Signaling Technology), anti-4EBP1 (9,452, Cell Signaling Technology), anti-Actin (3,700, Cell Signaling Technology), anti-Histone H3 (ab1791, Abcam), donkey Anti-Goat IgG H&L (FITC, ab6881), and goat anti-rabbit IgG H&L (HRP, ab6721) were purchased from Abcam. DAPI (d9542) and LPS (L6386) were purchased from Sigma Aldrich.

Anti-influenza A virus M2 monoclonal antibody (14C2; Abcam), anti-murine IL-12 (p70) polyclonal antibody (PeproTech), monoclonal anti-IL-12 antibody (Purified Rat Anti-Mouse IL-12 [p40/p70]; Becton, Dickinson and Company), and anti-HA (H1N1/California/06/2009) polyclonal antibody (Immune Technology Corp.) were purchased and used for Western blotting analysis and the FA assay. Antisera against swine IAH1, HKH5, and AnH7 viruses prepared from mice immunized with three doses (2,048–4,096 HA units) of purified viruses were also used for the detection of H5 or H7 HA proteins.
Antibodies against MPO (Agilent Technology) and Iba1 (Fujifilm Wako Pure Chemical Co.) were used for immunostaining for histological examination.
As secondary antibodies, goat anti-mouse IgG heavy and light chain cross-adsorbed antibody HRP-conjugated (Bethyl Laboratories, Inc.), goat anti-rabbit IgG H&L (Abcam), donkey anti-goat IgG H&L (HRP; Abcam), anti-mouse IgG (H + L) F(ab’)2 fragment (Alexa Fluor 488 conjugate; Cell Signaling Technology), and donkey anti-goat IgG H&L (Alexa Fluor 555; Abcam) were used. Anti-N2 NA monoclonal antibody specific to the influenza A virus was purchased from Tokyo Chemical Industry Company. To identify the N2 NA, anti-Aichi/2/68 (H3N2) virus serum was also used in the examination.