Alexa fluor 488 donkey anti goat igg h l

Following fixation with 4% paraformaldehyde, the oral mucosa around the upper maxillary molars was permeabilized and blocked with Blocking One (03953‐95; Nacalai Tesque, Kyoto, Japan). The sections were incubated with Anti‐Iba1 antibody (1:1000 dilution, ab5076; Abcam, Cambridge, UK) and anti‐inducible Nitric Oxide Synthase (iNOS) antibody (1:500 diluent, ab178945; Abcam) or with Anti‐Iba1 antibody (1:1000 dilution, ab5076; Abcam) and anti‐mannose (CD206) receptor antibody (1:500 diluent, ab64693; Abcam) overnight at 4°C. Following incubation, the sections were washed with phosphate‐buffer solution (PBS).
The secondary antibodies were Alexa Fluor 594 chicken anti‐rabbit IgG(H + L) (1:500; Invitrogen A21442) for anti‐iNOS antibody and anti‐mannose receptor antibody and Alexa Fluor 488 donkey anti‐goat IgG (H + L) (10 μg/ml; Invitrogen A11055, USA) for Anti‐Ibal1 antibody, and the sections were counterstained with 4′, 6‐diamidino‐2‐phenylindole (SJ217, Dojindo; Kumamoto, JP). The samples were observed under a fluorescence microscope (BZ‐9000; Keyence, Osaka, Japan). The images shown are representative of at least three separate experiments (Figure 2b).

Brains (700-µm thick block) and intestines (whole organ) were cleared with CLARITY protocol and then immunostained. Antibodies were used in this study - Primary antibody: Anti-GFAP (ab53554, Abcam, Cambridge, UK), Anti-beta III Tubulin antibody (ab18207, Abcam); Secondary antibody: Alexa Fluor 488 Donkey anti-Goat IgG (H + L) (A11055, Invitrogen, Carlsbad, CA), Alexa Fluor 488, F(ab’) 2-Goat anti-Rabbit IgG (H + L)(A11070, Invitrogen); Dyes: DyLight 594 labeled Lycopersicon Esculentum (Tomato) Lectin (DL-1177, Vector Labs, Burlingame, CA).
For immunostaining, the fixed sample was permeabilized in a pretreat solution, the mixture of 20% (w/v) DMSO (Sigma) and 2% (w/v) Triton X-100 (Sigma), in PBS at 37 °C for 4–5 h. After that, the sample was washed in PBS, transferred to a blocking solution, the mixture of 10% (w/v) BSA (bovine serum albumin; Bovogen, Australia) in a pretreat solution, and incubated at 37 °C for 4–5 h. After immunostaining with primary antibody (1:250 dilution) in PBS by rotating at 37 °C for 2–3 days, the stained samples were washed with PBS at RT for 1–2 h, then immunostained with secondary antibody (1:500 dilution) in PBS and incubated at 37 °C for 2–3 days. Stained samples were stored in PBS at 4 °C before clearing.

Tetramethylpyrazine hydrochloride (TMP) was obtained from Narula Research, LLC (Chapel Hill, NC, USA). The stock solution (100 mM) of TMP was prepared in water and kept at −20 °C. The stock is diluted in cultured media for in vitro experiments as and when required. Tris-base, glycine, sodium chloride, SDS, and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Alexa Fluor^® 594 donkey anti-mouse IgG (H + L) and Alexa Fluor^® 488 donkey anti-goat IgG (H + L) were procured from Life Technologies (Grand Island, NY, USA). Antibodies against MnSOD, fibronectin, vimentin, MMP-9, N-cadherin, α-tubulin, β-actin, and Lamin B were procured from Santa Cruz Biotechnology (Dallas, Texas, USA). Antibodies against p-PI3K (Tyr458), PI3K, p-Akt (Ser473), Akt, p-mTOR (Ser2448), mTOR, β-catenin, Wnt3a, p-GSK3β (Ser9), GSKβ, and FZD-1 antibodies were procured from Cell Signaling Technology (Danvers, MA, USA). An anti-p-GSK-3β antibody was procured from Abcam (Cambridge, UK). Forty-eight-well Chamber 3.2 mm diameter wells and 8 μm pores polycarbonate filters were purchased from Neuro Probe Inc (Gaithersburg, MD, USA).

The antibodies used in this work were: recombinant human CXCL13, human CXCL13 Quantikine ELISA Kit, mouse CXCL13 Quantikine ELISA Kit, mouse CXCL12 Quantikine ELISA Kit, mouse anti-human CXCR5-PE mAb, goat anti-human CXCL13 polyclonal Ab, goat anti-mouse CXCL13 polyclonal Ab (R&D, Minneapolis, MN), rat anti-mouse CD68-PE, rat anti-mouse CD45-APC, rat anti-mouse CD4-PerCP-CY5.5, rat anti-mouse CD19-FITC, rat anti-mouse CXCR5-PE/CY7 mAb (Biolegend, San Diego, CA), mouse anti-human CD68 mAb (Dako, Glostrup, Denmark), rabbit anti-mouse SPP1 polyclonal Ab (Proteintech, Chicago, IL), anti-TTF1 (Abcam, Cambridge, UK), rabbit anti-mouse E-Cadherin mAb, EMT Antibody Sampler Kit (Cell Signaling Technology, Beverly, MA), rabbit anti-human AhR polyclonal Ab, goat anti-human Lamin B polyclonal Ab, mouse anti-human ɑ-tubulin mAb (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human β-actin antibody (Sigma, St. Louis, MO), Alexa Fluor 488 Donkey anti-Goat IgG (H+L), Alexa Fluor 555 Donkey Anti-Mouse IgG (H+L), Alexa Fluor 647 Donkey anti-Rabbit IgG (H+L) (Life Technology, Thermo Fisher Scientific, Basingstoke, UK), and mouse SPP1 ELISA Kit (Shanghai GenePharma, Shanghai, China). BaP and DEX were purchased from Sigma.

All chemicals were purchased from Sigma-Aldrich Chemicals (St. Louis, MO) unless indicated differently. Tetramethylrhodamine methyl ester and Hoechst 33342 were from Life Technologies, USA. Primary goat anti-mouse polyclonal antibodies to vimentin (Abcam, USA) were paired with secondary AlexaFluor488 donkey anti-goat IgG (H+L), (Life Technologies, USA), and primary rabbit anti-mouse polyclonal antibodies to αSMA (Abcam, USA) were paired with secondary donkey anti-rabbit IgG (H+L) conjugated with AlexaFluor594 (Life Technologies, USA).