PAMs seeded in 12-well plates were infected with ASFV (MOI = 0.1) and fixed at 48 h post-infection with 4% paraformaldehyde (PFA) for 30 min at room temperature. Then the fixed cells were permeabilized with 0.2% Triton X-100 in PBS for 10 min at room temperature. 2% BSA in PBS was used to block the permeabilized cells for 3 h at room temperature. Anti-p30 antibody, anti-ASFV antibody positive serum, and the six generated anti-rpI215L mAbs were used, respectively, as primary antibodies to incubate the fixed cells for 2 h at room temperature. Coralite488-conjugated Affinipure Goat Anti-Mouse IgG(H + L) (Proteintech Group, Inc., Rosemont, IL, USA) was used as the secondary antibody to incubate the cells for 50 min at 37 °C. Five washes with PBS were performed after each of the above steps. Finally, the cells were observed and photographed by Zeiss Axio Observer (Carl Zeiss, Jena, Germany). Experiments with live ASFV were performed in the Biosafety Level 3 Laboratory of Lanzhou Veterinary Research Institute.
Coralite488 conjugated affinipure goat anti mouse igg h l
Manufactured by Proteintech
Sourced in United States
Coralite488-conjugated Affinipure Goat Anti-Mouse IgG(H + L) is a secondary antibody conjugated with the Coralite488 fluorophore. It is designed to detect and bind to mouse immunoglobulin G (IgG) antibodies in various immunoassay applications.
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Lab products found in correlation
Coralite594 conjugated goat anti rabbit igg h l, by Proteintech (5 mentions)
Axio observer, by Zeiss (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Goat anti mouse igg hrp, by Proteintech (1 mentions)
Superecl plus, by Advansta (1 mentions)
Hrp goat anti rabbit igg, by Proteintech (1 mentions)
Loperamide hydrochloride capsules, by Johnson & Johnson (1 mentions)
C met, by Proteintech (1 mentions)
Fibronectin, by Proteintech (1 mentions)
Anti phosphotyrosine antibody, by Abbkine (1 mentions)
E cadherin, by Proteintech (1 mentions)
Vimentin, by Proteintech (1 mentions)
Phospho akt ser473, by Cell Signaling Technology (1 mentions)
β actin, by Proteintech (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Beyotime (1 mentions)
Mg132, by Apexbio (1 mentions)
Cycloheximide (chx), by MedChemExpress (1 mentions)
Anti vegfa antibody, by Proteintech (1 mentions)
Anti neun, by Thermo Fisher Scientific (1 mentions)
Anti iba1, by Proteintech (1 mentions)
11 protocols using coralite488 conjugated affinipure goat anti mouse igg h l
1
Immunofluorescence Assay of ASFV Infection
2
Xuanhuang Runtong Tablets Characterization
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Xuanhuang Runtong tablets (batch number: 20200101) were purchased from Hunan Shidai Yangguang Pharmaceutical Co., Ltd. (Yongzhou, China). The voucher specimen is stored in the Chinese medicine storage cabinet of the Institute of Chinese Materia Medica, Hunan Academy of Traditional Chinese Medicine. Loperamide hydrochloride capsules (batch number: KFJ2P0P) were purchased from Xi'an Janssen Pharmaceutical Co., Ltd. (Xi'an, China). The reference substances aloin (batch number: 110787-201808, 92.4%), neohesperidin (batch number: 111857-201804, 99.4%), naringin (batch number: 110722-201805, 91.7%), echinacoside (batch number: 111670-201907, 91.8%), and aloe-epine (batch number: 110795-201007, 98%) were purchased from the China Institute for Food and Drug Control.
Alcian Blue and periodic acid Schiff stains were purchased from Visher Corporation (Changsha, China). Rabbit antimouse AQP3 primary antibodies were purchased from ABclonal (Wuhan, China). Cx43 and TLR5 primary antibodies, HRP goat antimouse IgG, HRP goat antirabbit IgG, and coraLite 488-conjugated affiniPure goat antimouse IgG (H + L) were purchased from Proteintech (Chicago, USA). SuperECL Plus was purchased from Advansta (California, USA). The IL-17A primary antibody was purchased from Abcam (Cambridge, UK). TLR5 and IL-17A primers were purchased from Shanghai Shenggong Biological Engineering Co., Ltd. (Shanghai, China).
Alcian Blue and periodic acid Schiff stains were purchased from Visher Corporation (Changsha, China). Rabbit antimouse AQP3 primary antibodies were purchased from ABclonal (Wuhan, China). Cx43 and TLR5 primary antibodies, HRP goat antimouse IgG, HRP goat antirabbit IgG, and coraLite 488-conjugated affiniPure goat antimouse IgG (H + L) were purchased from Proteintech (Chicago, USA). SuperECL Plus was purchased from Advansta (California, USA). The IL-17A primary antibody was purchased from Abcam (Cambridge, UK). TLR5 and IL-17A primers were purchased from Shanghai Shenggong Biological Engineering Co., Ltd. (Shanghai, China).
3
Protein Expression and Antibody Analysis
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Antibodies specific for c-Met (#25869-1-AP); c-Cbl (#25818-1-AP); Fibronectin (#1G10F9); Vimentin (#10366-1-AP); E-cadherin (#20874-1-AP); Tubulin (#10094-1-AP); DYKDDDDK (#66008-3-Ig); β-actin (#66009-1-Ig); mTOR (#20657-1-AP) were obtained from proteintech (Wuhan, China). The Phospho-AKT (Ser473) (#4060) and AKT (#9272) antibodies were both supplied by Cell Signaling Technology (Danvers, MA). An anti-phosphotyrosine antibody was obtained from Abbkine. Antibodies used were goat polyclonal to ORP5 (Abcam, ab59016); mouse monoclonal to N-cadherin (Servicebio, GB12135); p-mTOR (59. Ser 2448) (Santa Cruz Biotechnology, sc-293133). For immunoblotting, horseradish peroxidase-conjugated secondary antibodies were obtained from Beyotime. For immunofluorescence, CoraLite488––conjugated Affinipure Goat Anti-Mouse IgG(H + L) was obtained from proteintech.
Chloroquine Sulfate and MG-132 were from APExBIO (MA, USA). Cycloheximide (CHX) was from MedChemExpress (shanghai, China).
Chloroquine Sulfate and MG-132 were from APExBIO (MA, USA). Cycloheximide (CHX) was from MedChemExpress (shanghai, China).
4
Skin Wound Healing and Inflammation Analysis
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To evaluate the inflammation and epidermal regeneration in the wound area, tissues containing the wound site and their surrounding healthy skin were collected. The tissue samples were fixed in 4% (v/v) paraformaldehyde for 1 h right after sacrifice before embedded in paraffin. The samples were cross sectioned to slices (4 μm thickness) and then stained by Hematoxylin-Eosin (H&E). All slides were scanned and analyzed by a Digital Slide Scanner (KFBIO, Ningbo). The regenerated skins from the wound site were also excised for IF staining with Anti-VEGFA antibody (proteintech) and TNFA Ab (proteintech), respectively. CoraLite488-conjugated Affinipure Goat Anti-Mouse IgG(H + L) (proteintech) and CoraLite594-conjugated Goat Anti-Rabbit IgG(H + L) (proteintech) were used as the secondary antibody to reveal VEGFA and TNFA expression. The nuclei were stained with 4′,6-diamidino-2-phenylindole. Slides were observed under an upright fluorescence microscope (BX53, Olympus).
5
Immunohistochemical Analysis of Rat Brain
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After behavioral testing, the rats were anesthetized with an intraperitoneal injection of pentobarbital sodium. Following cardiac perfusion with 0.9% sodium chloride and 4% paraformaldehyde, the brains were extracted, fixed in 4% paraformaldehyde for 24 h and dehydrated using an alcohol gradient. Coronal paraffin sections with a thickness of 3 μm were obtained from paraffin blocks using a microtome. Subsequent to high-temperature repair and blocking, the sections underwent an overnight incubation at 4°C with the primary antibody, including anti-phospho-TAK1(Thr184/187) (rabbit. # PA5-99340; Thermo Fisher Scientific), anti-Neun (rabbit. # 26978-1-AP; Proteintech), anti-IBA1(mouse. # ab283319; Abcam), anti-glial fibrillary acidic protein (GFAP; mouse. # 60190-1-ig; Proteintech), and anti-GSDMD (rabbit. # 20770-1-AP; Proteintech). After three washes in phosphate buffer solution (PBS), the sections were exposed to appropriate conjugated secondary antibodies: CoraLite488-conjugated Affinipure Goat Anti-Mouse IgG([H+L] SA00013-1; Proteintech) and CoraLite594-conjugated Goat Anti-Rabbit IgG([H+L] #SA00013-4; Proteintech), followed by counterstaining with DAPI for 10 min in the dark. Images were captured using a fluorescence microscope.
6
Visualizing PAK5 and β-Catenin in Cells
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Seed post-treatment cells on Glass Bottom Culture Dishes (NEST, Wuxi, China) one day in advance. Cells were fixed with 4% paraformaldehyde for 20 min, and then blocked with TBS (0.3% Triton X-100 and 0.25% BSA) at room temperature for 2 h. Afterwards, they were incubated overnight at 4 °C with primary antibodies: anti-PAK5 (1:100, Abcam, Shanghai, China) and β-catenin (1:100, Santa Cruz, USA) Afterwards, they were washed three-fold with PBD. Afterwards they were stained with fluorescent secondary antibodies: CoraLite488–conjugated Affinipure Goat Anti-Mouse IgG(H + L) and CoraLite594–conjugated Goat Anti-Rabbit IgG(H + L) (1:200, proteintech, China) at room temperature for 60 min. Nuclei were deal with 4′, 6-Diamidino-2-phenylindole (DAPI) (KeyGen BioTECH) for 10 min. Pictures were taken by immunofluorescence confocal laser scanning microscopy (Zeiss LSM 880).
7
Autophagy Regulation by DHX15 Modulation
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Baf-A1 (s1413), Torin 1 (s2827), 3-MA (s2767), and Chloroquine diphosphate(CQ, s4157)were purchased from Selleck. The following antibodies were used: anti-LC3 (Sigma, L7645), anti-ACTB/β-actin (Cell Signaling, A5316), anti-phospho-p70 S6 kinase (Thr389) (Cell Signaling, 9234), anti-phospho-4E-BP1 (Thr37/46) (Cell Signaling, 2855), anti-SQSTM1/p62 (MBL PM045), anti-DHX15 (Abcam, ab70454), anti-Ki67 (Cell Signaling, 9449), anti-ULK1 (Cell Signaling, 8054), anti-phospho-ULK1(Ser757) (Cell Signaling, 14202), anti -p70 S6 kinase (Cell Signaling, 2708), anti-4E-BP1 (Cell Signaling, 9452) and CoraLite488–conjugated Affinipure goat anti-mouse IgG (H+L) (Proteintech, SA00013-1). DHX15-Flag was created in the CV702 by standard subcloning. DHX15 plasmid and its negative control were purchased from Genechem (Shanghai, China).
8
Immunostaining of NFATC1 and DYRK1A in U251 cells
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U251 cells were seeded in a glass‐bottom dish. When cells were 30%–50% confluent, immunostaining was performed as previously described.31 (link) The cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% PBST. Then, the cells were blocked with 5% BSA and successively incubated with primary and secondary antibodies in 5% BSA–0.1% PBST. Finally, the stained cells were mounted in VECTASHIELD mounting medium with DAPI (H1200, VECTOR Labs), and the images were captured with LIONHEART FX (BioTek). NFATC1 monoclonal antibody (MA3‐024, Thermo Fisher Scientific, Inc.) was used to detect NFATC1. DYRK1A polyclonal antibody (2771, Cell Signaling Technology, Inc.) was used to detect DYRK1A. Mouse IgG (Santa Cruz, sc‐2025) and rabbit IgG (Proteintech, B900610) were used as negative controls. CoraLite488—conjugated Affinipure goat anti‐Mouse IgG (H+L) (SA00013‐1, Proteintech, Wuhan, Hubei, P.R.C) and CoraLite594—conjugated goat anti‐rabbit IgG (H+L) (SA00013‐4, Proteintech) were used as secondary antibodies.
9
Immunofluorescence Analysis of Autophagy Markers
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The cells were inoculated on glass-bottomed Petri dishes and fixed with paraformaldehyde. After blocking, the cells were incubated overnight with primary antibody, and then incubated with secondary antibody for 90 min in the dark. The cells were photographed using the laser scanning confocal microscope (Leica SP8, Germany) after dyeing the nuclei with DAPI (1 μg/mL). Primary antibodies included P62 (1:200; Abcam ab205719), LAMP1 (1:200; Santa Cruz Biotechnology H4A3), LC3B (1:200; Sigma-Aldrich L7543); secondary antibodies included anti-mouse secondary anti Cy3-conjugated Affinipure goat anti-mouse IgG (H + L) (1:200; Proteintech SA00009-1), CoraLite488-conjugated Affinipure goat anti-mouse IgG (H + L) (1:200; Proteintech SA00013-1), CoraLite594-conjugated Affinipure goat anti-rabbit IgG (H + L) (1:200; Proteintech SA00013-4). For LysoTracker (Beyotime, Shanghai, CHN, C1046), we also inoculated the cells on glass-bottomed Petri dishes. After growing to a suitable density, cell culture fluid containing a LysoTracker final concentration of 75 nM was added to the cells, which were photographed after 45 m of stimulation. Use ImageJ to count the number of fluorescent dots and the Pearson correlation coefficient.
10
Indirect Immunofluorescent Assay for ASFV
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Indirect Immunofluorescent Assay (IFA) was conducted on 4% PFA-treated ASFV-infected PAMs (a kind gift provided by Lanzhou Veterinary Research Institute). PAMs were collected from 4-week-old pigs and seeded onto 96-well plates, incubated in RPMI 1640 medium (Gibco, Thermo Scientific, Waltham, MA, USA) with 10% FBS (Gibco, Thermo Scientific, Waltham, MA, USA) at 37 °C with 5% CO2. PAMs were infected with ASFV (MOI = 0.1) for 48 h before the cells were fixed with 4% PFA in PBS for 30 min at room temperature and then stored at 4 °C. Experiments with live ASFV were performed in the Biosafety Level 3 Laboratory of Lanzhou Veterinary Research Institute. The fixed cells were incubated with anti-rp54 mAb before incubation with Coralite488-conjugated Affinipure Goat Anti-Mouse IgG(H+L) (Proteintech Group, Inc., Rosemont, IL, USA), and the plates were examined by Zeiss Axio Observer.
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