Alexa fluor 488 goat anti rabbit igg a11034

Antibodies against the neurofilament (NF, C28E10) and synapsin (SYN, D12G5) were purchased from Cell Signaling Technology. The antibody against S100β (IR504) was purchased from Dako. CF568 α-bungarotoxin (#00006, 1:3000 for staining) was purchased from Biotium. CD68 (D4B9C) was purchased from Cell Signaling. AlexaFluor-488 goat anti-rabbit IgG (A-11034) and AlexaFluor-488 goat anti-mouse IgG (A-11001) were purchased from Invitrogen. β-galactosidase (β-Gal, SAB3500043) was purchased from Sigma. The remaining chemicals were purchased from Sigma-Aldrich unless otherwise stated. SYBR green mix (100029284) was purchased from Takara. A high-capacity cDNA Reverse Transcription kit (4368814) was purchased from ABI. In addition, μ-conotoxin GIIIB (H-9015.1000BA) was purchased from Bachem Americas.

The makers for the quality control of MFX with ephedrine hydrochloride and pseudoephedrine hydrochloride were purchased from National Institutes for Food and Drug Control (Beijing, China), and benzoylaconine, benzoylmesaconine, benzoylhypaconine, and asarinin were purchased from Chengdu Mansite Pharmaceutical Co., Ltd. (Sichuan, China). Escitalopram was purchased from Dalian Meilun Biotechnology Co., Ltd. (Liaoning, China). Lipopolysaccharide (LPS) was bought from Sigma-Aldrich Co., Ltd. (St. Louis, USA). Rabbit polyclonal anti-NLRP3 (ab214185), rabbit polyclonal anti-IL-1β (ab9722), rabbit monoclonal anti-TXNIP (ab188865), rabbit monoclonal anti-DCX (ab207175), rabbit polyclonal anti-DCX (ab18723), and rabbit monoclonal anti-BDNF (ab108319) were acquired from Abcam. Rabbit monoclonal anti-ASC (67824S) and rabbit monoclonal anti-TrkB (4603T) were offered by Cell Signaling Technology. Mouse monoclonal anti-caspase-1 (sc-56036) was obtained from Santa Cruz Biotechnology. Goat polyclonal anti-IL-1β (AF-401-NA) was purchased from R&D. Rabbit polyclonal anti-β-tubulin (abs131994) and rabbit polyclonal anti-GAPDH (abs132004) were obtained from Absin Bioscience Inc. Alexa Fluor 488 Goat anti-Rabbit IgG (A-11034) and Alexa Fluor 594 Rabbit anti-Goat IgG (A27016) were purchased from Invitrogen.

Spermatocyte chromosome spreads were treated with RNaseT1 (EN0541; Thermo Fisher Scientific), 1:200 dilution in staining buffer PBS with 0.1% Tween supplemented with 3 mM MgCl₂ by incubation at 37°C for 1 h (protocol adapted from Smolka et al [2021] (link)). After washing with staining buffer, the spreads were treated with RNaseH in RNaseH Reaction buffer (10 units per 100 μl of reaction mix; M0297L; New England Biolabs) for another 1 h at 37°C. Treatment of spreads with only RNaseT1 (same dilution as mentioned before) and only RNaseH (same concentration as before) was performed for 1 h at 37°C. Subsequent to washing and blocking with 10% goat serum, the spermatocytes were primary immunolabelled with 1:100 dilutions of clone S9.6 mouse monoclonal antibody (MABE1095; Merck Sigma-Aldrich) and rabbit anti-SYCP3 (NB00-230; Novus). Anti-rRNA staining was done with 1:100 dilutions of anti rRNA Y10b (sc-33678; Santa Cruz Biotechnology). In all cases, primary antibody incubation was performed overnight at 4°C; the slides were then washed and stained with 1:1,000 dilution of secondary antibodies (Alexa Fluor 488 goat anti rabbit IgG, A-11034; Invitrogen and Alexa Fluor 568 goat anti mouse IgG, A-11004; Invitrogen). After washing with staining buffer, the slides were mounted with Vectashield containing 1 μg/ml of DAPI.

Rabbit polyclonal antibodies to phospho-PKA substrate (9621S), PKA C-α (4782S), phospho-PFK2 (13064S), phospho-Ser16/Thr17-phospholamban (8496S), and phospho-Troponin I were purchased from Cell Signaling Technology. Rabbit polyclonal anti-PDE4D (ab14613) was purchased from Abcam. Alexa Fluor 488 goat anti-rabbit IgG (A11034) and Alexa Fluor 546 phalloidin (A22283) were purchase from Invitrogen. Insulin solution human (19278), (-)-Isoproterenol hydrochloride (16504), 3-Isobutyl-1-methylxanthine (15879) and 8-Bromoadenosine 3’,5’-cyclic monophosphate sodium salt (B7880) were purchased from Sigma. Phosphodiesterase inhibitor Tocriset containing Milrinone, Cilostamide, Zardaverine, (R)-(-)-Rolipram, and Ro 20–1724 (Cat. No. 1881) along with MMPX (Cat. No. 0552) and EHNA hydrochloride (Cat. No. 1261) were purchased from Tocris.

LX2 cells were grown on glass coverslips and treated as described in the Results and figure legends. Then, cells were washed twice with phosphate buffered saline (PBS), fixed in 4% paraformaldehyde for 10 min and processed for immunofluorescence with the indicated primary antibodies: anti-αSMA (A-2547) purchased from Sigma-Aldrich, anti-COL1A1 (sc-8784) and anti-MMP9 (sc-6840) from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and the appropriate FITC-conjugated secondary antibodies [Alexa Fluor^® 488 goat anti-rabbit IgG (A11034) or Alexa Fluor^® 488 goat anti-mouse IgG (A11001), Life Technologies, Grand Island, NY, USA]. Mounting medium used was Fluoromont G^® (BioNova cientifica, Madrid, Spain). Immunofluorescence was examined using a confocal microscope (Leica TCS SP5 X, Barcelona, Spain) and image analysis procedures were performed with Fiji software (NIH, Bethesda, MD).

Cells were infected, fixed (3.7% formaldehyde, 10 min), permeabilized (0.25% Triton X-100, 5% FBS, 15 min), blocked (5% FBS, 1h at RT), incubated with primary antibodies in 5% FBS overnight at 4°C (anti-IL-1 beta, 1:100, ab9722; anti-MMP7, 1:25, ab4044, all Abcam; anti-ASC, 1:50, sc-22514-R, Santa Cruz; anti-NLRP3/Cryo-2, 1:100, AG-20B-0014-C100, Adipogen) and appropriate secondary antibody (Alexa Fluor 488 goat anti-rabbit IgG, A-11034, or goat anti-mouse IgG, A-11001; Life Technologies), (1h at RT). After nuclear staining (DRAQ5, Abcam), slides were mounted (Fluoromount, Sigma-Aldrich), imaged by laser-scanning confocal microscopy (LSM510 META confocal microscope, Carl Zeiss) and quantified by ImageJ software 1.46r (NIH).