Ab25088

Aliquots of total kidney homogenate from individual animals were diluted in lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 % Triton X-100, 0.1 % SDS, 2 mM EDTA, 0.1 mM EGTA, 5 mM NaF, 1 mM Na₃VO₄, 5 mM Na₂PO₄ and 1 × proteinase inhibitor cocktail (Beyotime Institute of Biotechnology, China) to a final protein concentration of 2 μg/μL. Western blot analysis was conducted and quantified as described by Müller et al. [28 (link)]. The following antibodies were used: rabbit anti-FKN antibody (ab25088, Abcam Ltd, Hong Kong, 1:100 dilution) or anti-NFκB p65 antibody (ab31481, Abcam Ltd, Hong Kong, 1:200 dilution) or anti-NF-κB phospho p65 antibody (ab28810, Abcam Ltd, Hong Kong, 1:100 dilution) to evaluate the activity of NF-κB pathway, and mouse anti-beta actin monoclonal antibody (AA128, Beyotime Institute of Biotechnology, China, 1:500 dilution) followed by the goat polyclonal secondary antibody to rabbit IgG-H&L (HRP) (ab6721, Abcam Ltd, Hong Kong, 1:1000 dilution) or anti-mouse IgG-H + L (A0216, Beyotime Institute of Biotechnology, China, 1:1000 dilution). The image of western blots was scanned by Quantity One software and the original intensity of each specific band was quantified with freeware image analysis software, NIH Image (National Institute of Health, Bethesda, Md., USA).

Rabbit polyclonal antibody cx3cl1: model (ab25088) and rabbit polyclonal antibody CCL28: model (ab232837) were purchased from Abcam, Cambridge, UK. All the specimens were fixed in formalin and made into paraffin sections by the routine process. All the sections were fully baked for immunohistochemical staining. PBS instead of the primary antibody was used as the negative control, known positive sections were used as the positive control, and the ratio of cell staining intensity to the number of positive cells was used for the comprehensive score.
The cytoplasm from light yellow to dark brown was used as the marker of positive cells, which was 5 times higher (× 200) count the positive cells in the visual field, and calculate the positive rate: 0–5% is 0, 6%–25% is 1, 26%–50% is 2, 51%–75% is 3, 76%–100% is 4. According to the staining conditions of most cells, the staining intensity standard is set, including 3 points for tan, 2 points for brown, 1 point for light yellow, and 0 points for nonstaining. Finally, the product of positive cell percentage and staining intensity score was calculated, and the total score was (-): 0; (+): 1–4 points; (++): 5–8 points; (++ +): 9–12 points. Score ≥5 was defined as medium/high positive expression of cx3cl1 or CCL28 in tumor cells.

Anti-CX3CL1 antibody (ab25088), anti-CX3CR1 antibody (ab8021), anti-CX3CL1 antibody (ab25088), anti-CX3CR1 antibody (ab8020), anti-CD45 antibody (ab8216), anti-MHC class II antibody (ab23990), anti-Runx1 antibody (ab23980), anti-Iba1 antibody (ab5076), anti-NSE antibody (ab53025), and anti-NeuN (ab104224) were from Abcam (USA). Anti-MCPIP antibody (sc-515275) and β-tubulin (sc-9014) were obtained from Santa Cruz Biotechnology (USA). Anti-C/EBPα antibody (2295), anti-rabbit-IgG-HRP (7074s), and anti-mouse-IgG-HRP (7076s) were obtained from the Cell Signaling Technology (USA). Alexa Fluor-488 donkey anti-rabbit IgG antibody (A21206), Alexa Fluor-555 donkey anti-mouse IgG antibody (A31570), Alexa Fluor-555 donkey anti-goat IgG antibody (A21432), and Alexa Fluor-633 donkey anti-goat IgG antibody (A21082) were from the Life Technologies. RBFOX3/NeuN antibody [Alexa Fluor-405] (NBP1-77686AF405) was from Novus (USA).

Frozen hippocampi of untreated WT and GABA/CB1^−/− mice were lysed in 1% SDS buffer (Sigma-Aldrich, Munich, Germany) containing protease inhibitor (Complete Mini, Roche), sonicated and clarified by centrifugation (13,000 rpm for 10 min). Protein concentrations were determined using BCA Protein Assay Kit (Pierce). Equal amounts of protein were run on NuPAGE Bis-Tris 4%–12% gradient gels (Invitrogen, Carlsbad, CA, USA). In preliminary experiments we determined the range where the relation between the protein concentration and signal intensity is linear for each antibody. The proteins were subsequently blotted onto PVDF-membranes using the iBlot Dry Blotting System (Invitrogen, Carlsbad, CA, USA). The blots were incubated with primary antibodies to CD200 (bs-6030R; 1:1,000; Bioss Antibodies), CD200R (1:1,000; ab34097; Abcam), CX3CL1 (1:1,000; ab25088; Abcam), CX3CR1 (1:1,000; C8354; Sigma-Aldrich), Sirpa (1:1,000; ab53721; Abcam), CD47 (1:1,000; sc25773; Santa Cruz) and with an antibody against GAPDH (1:12,000; ab9484; Abcam). The blots were then incubated with peroxidase-conjugated secondary antibodies, followed by the ECL substrate (Pierce). Images were created using the ChemiDoc Imaging System (Bio-Rad Laboratories) and the quantification was performed using the ImageLab software (Bio-Rad Laboratories). Signal intensities were normalized to GAPDH.

For immunohistochemistry (IHC), formalin-fixed and paraffin embedded renal sections were prepared as described previously, and then incubated in 3 % hydrogen peroxide for 15 min and normal goat serum for 10 min to block endogenous peroxidase. The sections were incubated with either anti-FKN antibody (ab25088, Abcam Ltd, Hong Kong, 1:200 dilution) or anti-NFκB p65 antibody (ab31481, Abcam Ltd, Hong Kong, 1:100 dilution) overnight at 4 °C and then incubated with goat polyclonal secondary antibody to rabbit IgG-H&L (HRP) (ab6721, Abcam Ltd, Hong Kong, 1:500 dilution) for 30 min at 37 °C. Normal rabbit serum served as a negative control. To compare the expression levels of FKN and p65 in glomerular cells by IHC, staining intensity was evaluated semiquantitatively according to the previous paper [10 (link)]. Forty or more glomeruli were examined on each slide and assigned values of staining intensity from 0 to 3+. An intensity score was calculated as: (% glomeruli intensity (negative) × 0) + (% glomeruli intensity (trace intensity) × 0.5) + (% glomeruli (1+) intensity × 1) + (% glomeruli (2+) intensity × 2) + (% glomeruli (3+) intensity × 3). The values typically ranged from 0 to a maximum of 300.

Total protein was extracted from the hippocampus of rats or primary cultured neurons for immunoblotting analysis. The protein concentrations of all extracted samples were measured using the Bio-Rad Protein Assay (BioRad, Hercules, CA) with bovine serum albumin (BSA) standards. Fifty micrograms of the protein samples was separated by SDS-PAGE and then transferred to PVDF membranes, which were then incubated with primary antibodies at 4 °C overnight followed by fluorescent secondary antibodies (LICOR Biosciences, Lincoln, NE, USA). Anti-CX3CL1 (1:1000, Cat. #ab25088, Abcam, USA) and anti-CX3CR1 (1:1000, Cat. #ab8021, Abcam, USA) were used as the primary antibodies. β-Actin (1:1000, Cat. #G8795, Sigma, St. Louis, MO, USA) was used as an internal control. The bands on the blot were detected with the Odyssey infrared imaging system (LICOR Biosciences, Lincoln, USA). The signal intensities were analyzed using Odyssey v. 1.2 software and normalized to the intensity of the loading control, β-Actin.