Ab66579

Mice were decapitated 24 hr after the behavioral tests, and brains were rapidly removed. According to the mouse brain map (Paxinos & Franklin, 2004), the hippocampus was dissected on ice and placed into liquid nitrogen to be frozen and then stored at −80°C until use. Proteins from hippocampus were dissolved in RIPA lysis buffer (CW Biotech), and their concentration was measured using BCA Protein Assay Kit (CW Biotech). Total proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) in reducing conditions using 12% or 8% acrylamide gels and analyzed by immunoblotting using anti‐TLR4 (1:1,000; ab13867; Abcam), anti‐TNF‐α (1:1,000; ab66579; Abcam), and anti‐β‐actin (1:10,000, #3700; CST) as primary antibodies. Band intensity was semi‐quantitatively analyzed using the Quantity One software (Bio‐Rad), and the expression level of TLR4 and TNF‐α was normalized to that of β‐actin.

A subset of 6 mice per group were used for assessment of hypothalamic PERK, pPERK^Thr980, eIF2α, peIF2α^Ser55, IRE1α, pIRE1α^Ser724, ATF6, TNFα and tubulin protein expression using standard methodology, as previously described (McGavigan et al., 2015 (link)). Antibodies against PERK, pPERK^Thr980 and peIF2α^Ser55 were from Cell Signaling (3179 and 3398, respectively). Antibodies against eIF2α, ATF6 and tubulin were from Santa Cruz (sc-133227, sc-22799, sc-23948, respectively) and TNFα, IRE1α and pIRE1α^Ser724 antibodies were from Abcam (ab66579, ab37073, ab104157, respectively). All antibodies were used at a concentration of 1:1000 and pixel densities of immunoreactive bands were quantified using FluorChem 9900 (Alpha Innotech, CA).

Immunoblotting was performed as previously described [16 (link), 18 (link)]. Primary antibodies were obtained from Santa Cruz Biotechnology: α tubulin, 1:10000, sc-23948; BiP, 1:1000, sc-13968; XBP1, 1:1000, sc-7160; Akt, 1:5000, sc-8312; Cell Signaling Technology: pAkt, 1:5000, S473 or Abcam: IRE, 1:2000, ab37073; pIRE, 1:1000, ab48187; TNFα 1:5000 ab66579. Chemiluminescence was performed with Luminata Forte Western HRP Substrate (Millipore) and detected using Chemidoc MP Imaging system. Protein expression was quantified using ImageLab 5.1 software.

After treatment, cells were washed with ice-cold PBS and lysed in lysis buffer. The protein concentration of each sample was measured using a NanoPhotometer N60 (Implen, Germany). The samples were separated on 6%, 8%, or 10% (v/v) sodium dodecyl sulfate polyacrylamide gels and then transferred onto polyvinylidene difluoride membranes. After blocking with 5% dehydrated milk for 1 h, the membranes were incubated with the following primary antibodies at 4 °C overnight: TNF-α (ab66579, 1:1000, Abcam), iNOS (13,120, 1:1000; CST), and GAPDH (5174, 1:1000, CST). The membranes were then washed three times with TBST and incubated with secondary antibodies (anti-rabbit or anti-mouse) for 1 h at room temperature. Finally, chemiluminescence was developed using enhanced chemiluminescence reagents.

Rats were perfused with 10 mM phosphate-buffered saline (PBS) (pH 7.4). Subsequently, the perfusion fluid was replaced by 4% phosphate-buffered paraformaldehyde (4% PFA). The heart was excised and subsequently fixed in 4% PFA for 24 hours following which the tissue was cryopreserved in 4% PFA containing 30% sucrose at 4°C. Cryostat sections (40μm) were obtained and washed (three times) in 10 mM PBS (pH 7.4). Non-specific protein interaction was blocked by incubating sections in rabbit serum prepared in 10mM PBS (with 0.01% Triton X-100) for 2 hours. Sections were washed 3 times with PBS and incubated with primary antibody solution overnight at 4°C. Primary antibodies against TNF-α (ab66579), IL-1β (ab2105) and MMP-9 (ab38898) were obtained from Abcam and prepared in 10 mM PBS (with 0.03% Triton X-100 and normal serum). The sections were then incubated in a solution of secondary antibody [Goat anti-Rabbit (1:1000; Thermo Fisher Scientific, Waltham, MA, USA), 10 mM PBS, 0.03% Triton X-100 and normal serum] for 1 h in a dark enclosure at room temperature. Immuno-labeled sections were washed and mounted on gelatin-coated slides with plain anti-fade mounting medium containing 4’,6-diamidino-2-phenylindole (DAPI) (Vector Labs). The immunostained sections were imaged using an Olympus FluoView 10i microscope (Olympus America, Center Valley, PA, USA).

For immunofluorescence (IF) staining, sectioned slides (n=3 per group) were randomly chosen. At first, all slides were deparaffined with xylene and hydrated with graded ethanol and distilled water. Then, all slides were blocked with 5% (wt/v) bovine serum albumin (BSA, Sangon Biotech, China) solutions at RT for 1 h. The blocked slides were then incubated with primary rabbit/mouse specific antibodies (α-SMA (Abcam, ab32575), CD31 (Abcam, ab222783), IL-1β (Abcam, ab254360), and TNF-α (Abcam, ab66579) at 4°C overnight. Then, the slides were incubated using Alexa Fluor^® 488 or 546 (1:500, Invitrogen) conjugated secondary antibodies at RT for 2h to visualize these proteins.