Paraffin sections of liver were deparaffinized with a deparaffinizing solution (G1128; Servicebio, Wuhan, China), rehydrated with different concentrations of alcohol, and washed in PBS (G0002; Servicebio, Wuhan, China) three times. The sections were incubated in 3% H2O2 solution at room temperature for 25 min in dark conditions to block endogenous peroxidase, blocked with a rapid closure solution at room temperature for 30 min, and finally sequentially incubated with primary antibodies at 4 °C overnight and secondary antibodies at room temperature for 50 min. Sections were developed using diaminobenzidine color-developing solution (G1212; Servicebio, Wuhan, China) and re-stained with hematoxylin (G1004; Servicebio, Wuhan, China). Sections were observed and photographed using a light microscope (Nikon E100; Nikon Corporation, Tokyo, Japan). Information on all the antibodies is shown in Table S2 .
G0002
Manufactured by Wuhan Servicebio Technology
Sourced in United States
The G0002 is a centrifuge designed for general laboratory use. It is capable of separating samples at high speeds to facilitate various scientific procedures. The core function of the G0002 is to provide a controlled and consistent centrifugal force to separate components within a sample based on their density differences.
Automatically generated - may contain errors
Lab products found in correlation
G5001, by Wuhan Servicebio Technology (2 mentions)
G1012, by Wuhan Servicebio Technology (2 mentions)
G1401, by Wuhan Servicebio Technology (2 mentions)
G1128, by Wuhan Servicebio Technology (1 mentions)
G1212, by Wuhan Servicebio Technology (1 mentions)
G1004, by Wuhan Servicebio Technology (1 mentions)
G1202, by Wuhan Servicebio Technology (1 mentions)
Tsy b, by Wuhan Servicebio Technology (1 mentions)
Series s sensor chip, by GE Healthcare (1 mentions)
Biacore 8k, by GE Healthcare (1 mentions)
Gb11130, by Wuhan Servicebio Technology (1 mentions)
Gb11022 3, by Wuhan Servicebio Technology (1 mentions)
Gb13044, by Wuhan Servicebio Technology (1 mentions)
Gb11179, by Wuhan Servicebio Technology (1 mentions)
Collagenase type 2, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Wuhan Servicebio Technology (1 mentions)
Af0120, by Affinity Biosciences (1 mentions)
Af6139, by Affinity Biosciences (1 mentions)
Eclipse c1, by Nikon (1 mentions)
Citrate antigen retrieval solution, by Solarbio (1 mentions)
13 protocols using g0002
1
Immunohistochemical Analysis of Liver Tissue
2
Harvesting and Preserving Organs from ApoE-/- Mice
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Briefly, after each mouse was weighed, 10% chloral hydrate solution was used to anesthetise the ApoE−/− mice (0.004 ml/g body weight) through intraperitoneal injection. The mice were then fixed in a supine position with their necks extended. A median incision to the abdomen was made to open the abdominal cavity and then the thoracic cavity to harvest organs in the following sequence: liver, kidney and heart. Organs were fixed in 4% buffered paraformaldehyde solution, embedded in paraffin using paraffin embedding machine JB‐P5 (Junjie Electro Co., Wuhan, China) and archived. Then, 3‐μm thick sample tissue sections were prepared using pathology slicer RM2016 (Laika Alliance Co. of Shanghai, USA). The sections were sequentially washed in triplicate in xylene (15 min each), dehydrated twice in pure ethanol (5 min each), and dehydrated in an ethanol gradient of 85% and 75% (5 min each). The sections were incubated in sodium citrate antigen retrieval solution (Servicebio: G1202, pH 6.0) and washed 3 times with PBS (Servicebio: G0002, pH 7.4) on a rocker device (Servicebio: TSY‐B, Germany) for 5 min per wash. Objective tissues were marked with a liquid blocker pen (Gene tech: GT1001, China) and soaked in 3% BSA (Servicebio: G5001, China) at room temperature for 30 min.
3
TUNEL Assay for Apoptosis Detection
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Cell slides fixed with 4% paraformaldehyde (DF0135, Leagene Biotechnology Co., Ltd., Beijing, China) were washed with Phosphate-Buffered Saline (PBS) (G0002, Servicebio Biotechnology, Wuhan, China). A suitable amount of 0.3% Triton X-100 permeabilization solution (T795, Beyotime Biotechnology, Shanghai, China) was added to the slides. Subsequently, TUNEL staining was performed according to the instructions of the one-step TUNEL apoptosis detection kit (FITC green fluorescence) (G1501, Sevier Biotechnology Co., Ltd., Wuhan, China). Detection solution (TdT enzyme: fluorescent labeling solution= 1:9) was added by drops, and the samples were incubated at 37 °C for 1 hour in the dark. After one cycle of PBS washing, 4ʹ,6-diamidino-2-phenylindole (DAPI) was added in drops to stain for 5 minutes, and slides were dehydrated and mounted with neutral resin (10004160, Sinopharm, Beijing, China). Image acquisition of the slices was conducted using a Mshot inverted microscope (MF53, Guangzhou Mingmei Photoelectric Technology Co., Ltd., Guangzhou, China). The nuclei of apoptotic cells emitted green fluorescence, while the DAPI-stained nuclei emitted blue fluorescence.
4
Measuring Inhibition and Binding of ATG4B
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The inhibition of compounds on ATG4B activity was performed as our previous report [36 (link)]. The percentage of inhibition was used to plot drug concentration-response curve and calculate IC50 values (Graphpad 8.0, GraphPad Software, La Jolla, CA, USA). The binding affinity of Am-F4a and ATG4B protein was measured by SPR assay on a Biacore 8 K instrument (GE Healthcare, Piscataway, NJ, USA). Briefly, purified ATG4B protein (200 μg/mL, pH 8.0) were immobilized (~10,000RU) on a Series S Sensor Chip (GE Healthcare, Piscataway, NJ, USA) according to a standard amine coupling procedure. Running buffer for immobilization was PBS (Servicebio, G0002, pH7.2-7.4) containing 5% DMSO. After immobilization, compound Am-F4a serially diluted in running buffer was as stock solution. Seven concentrations of Am-F4a (20, 10, 5, 2.5, 1.25, 0.625, 0 μM) were simultaneously injected at a flow rate of 65 μL/min for 60 s of association phase at 25 °C. Biacore 8K manager software was used to calculate the equilibrium dissociation constant (Kd).
5
Immunohistochemical Analysis of Liver Fibrosis
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Before immunostaining, paraffin-embedded sections were dewaxed and rehydrated in an alcohol series. The sections were washed with phosphate-buffered saline after heat-induced antigen retrieval. Next, the sections were treated with 3% hydrogen peroxide for 15 m to eliminate endogenous peroxidase activity and then incubated with 3% bovine serum albumin for 30 m at 37°C to block non-specific binding sites. Subsequently, sections were incubated overnight at 4°C with diluted primary antibodies, including α-SMA (1:2,000; GB13044; Servicebio, Inc. Wuhan, China), collagen I (1:1,200; GB11022-3; Servicebio, Inc.), matrix metalloproteinase (MMP)-2 (1:1,200; GB11130; Servicebio, Inc.), tissue inhibitors of metalloproteinase-1 (TIMP-1) (1:1,000; 106164-T40; Sino Biological US, Chesterbrook, PA, USA), TGF-β1 (1:500; GB11179; Servicebio, Inc.), and incubated with a conjugated secondary antibody after washing with phosphate-buffered saline (G0002; Servicebio, Inc.). Diaminobenzidine kits (G1211; Servicebio, Inc.) were used to visualize antibody binding areas. For each mouse, we analyzed three liver slices and randomly observed the positive-stained areas in three different fields of vision on each slice. The ratio of positive-stained to total area was calculated by Image-Pro Plus 6.0.
6
Isolation and Culture of Degenerated Intervertebral Disc Cells
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The HNP cells were isolated in vitro from Pfirrmann Grade II patients. Briefly, the NP tissues obtained intraoperatively were transported to ultra-clean laboratory with 0.9% sodium chloride solution. After being washed 3 times with sterilized PBS (G0002, Servicebio, China), the NP tissues were digested with 0.25% Trypsin-EDTA (G4001, Servicebio, China) for half an hour, and with equal amount of collagenase type II (0.2%, Invitrogen, USA) and complete DMED/F-12 medium (contained 10% fetal bovine serum and 1% penicillin-streptomycin) for another 1 h under a shaker (37 °C, 75 rpm). After being centrifuged with 1250 rpm for 5 min, HNP were resuspended with complete DMED/F-12 medium (contained 10% fetal bovine serum and 1% penicillin-streptomycin). Subsequently, the HNP cells were counted and replanted at T25 culture flask in an aseptic atmosphere of 5% CO2 at 37 °C. The HEK 293 T cells were purchased from QuiCell Biotechnology Co., LTD (Shanghai, China). Further experiments were available when the confluence reached to 80%. To simulate the role of oxidative stress and cell dysfunction during IVDD, cells were exposed to tert-butyl hydroperoxide (TBHP) at the concentration of 100 μM for 3 h, at which time TBHP media were replaced with fresh media.
7
Immunofluorescence Analysis of Apoptosis Markers
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The intestinal tissues were made into paraffin section, and dewaxed with xylene, then ethanol was successively added with high to low concentration to rehydrate the tissue, and antigen repair solution was added, then the sections were incubated in 5% BSA (G5001, SERVICEBIO), 0.2% Triton X-100 and PBS (G0002, SERVICEBIO) to permeabilize the tissue sections. Followed, the sections were incubated with the BAX (AF0120, 1 : 200, Affinity) and Bcl-2 (AF6139, 1 : 200, Affinity) overnight at 4°C, then incubated with Goat Anti-Rabbit IgG (H + L) Fluor488-conjugated (S0018, Affinity) at room temperature for 0.5 hours. Then washed away the antibody, and the DAPI (G1012, SERVICEBIO) was added to stain the cell nuclear. The NIKON ECLIPSE C1 was used to observe and take pictures. ImageJ software was used to analyze the BAX and Bcl-2 protein fluorescence intensity.
8
Cell Cycle Synchronization by Double Thymidine Blocking
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Cells were synchronized at the G1/S boundary by the standard double thymidine blocking method. Briefly, cells were first blocked with thymidine (HY‐N1150, MCE, 2 × 10−3m ) for 16 h, washed with phosphate buffered saline (PBS, G0002, Servicebio) twice, released in fresh media for 9 h, and then exposed to thymidine (2 × 10−3m ) for an additional 16 h. Synchronized cells were released into fresh media and collected at the indicated time points for cell cycle and Western blot analysis.
9
Immunofluorescent Detection of UCP1 in Paraffinized Tissues
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All tissues were fixed in 4% paraformaldehyde over 24 h at 4°C, then dehydrated and embedded into paraffin.
After that, tissues were sectioned into thick slices (4 to 6 µm) and stained with hematoxyline and eosin (H&E).
For immunofluorescence, slices were first boiled for 15min in Citrate Antigen Retrieval solution (C1032; Solarbio) diluted with double distilled water to pH 6.0. After cooling to room temperature, we circled the tissue with a PAP pen (ab2601; Abcam), dropped 3% bovine serum albumin (GC305006; Servicebio) in PBS (G0002; Servicebio), which covered the tissue and it then got blocked for 30min. The slices were incubated with rabbit polyclonal anti-UCP1 primary antibody (23673-1-AP; Proteintech) 1:50 diluted in PBS overnight at 4°C. The slices were rinsed in PBS for 3 times, followed by treating with Goat Anti-Rabbit IgG H&L/Alexa Fluor 594 (bs-0295G-AF594; Bioss) incubated at room temperature for 50 min. Every move since this step needs to avoid light. DAPI (C1002; Beyotime) stained uncleus for 10 min and the slices were washed in PBS for 3 times and sealed with anti-fade mounting medium (G1401; Servicebio) for longer storage. Photomicrographs were taken under a confocal laser-scanning microscope (Leica; Germany). Images shown are representative results of at least three biological replicates.
After that, tissues were sectioned into thick slices (4 to 6 µm) and stained with hematoxyline and eosin (H&E).
For immunofluorescence, slices were first boiled for 15min in Citrate Antigen Retrieval solution (C1032; Solarbio) diluted with double distilled water to pH 6.0. After cooling to room temperature, we circled the tissue with a PAP pen (ab2601; Abcam), dropped 3% bovine serum albumin (GC305006; Servicebio) in PBS (G0002; Servicebio), which covered the tissue and it then got blocked for 30min. The slices were incubated with rabbit polyclonal anti-UCP1 primary antibody (23673-1-AP; Proteintech) 1:50 diluted in PBS overnight at 4°C. The slices were rinsed in PBS for 3 times, followed by treating with Goat Anti-Rabbit IgG H&L/Alexa Fluor 594 (bs-0295G-AF594; Bioss) incubated at room temperature for 50 min. Every move since this step needs to avoid light. DAPI (C1002; Beyotime) stained uncleus for 10 min and the slices were washed in PBS for 3 times and sealed with anti-fade mounting medium (G1401; Servicebio) for longer storage. Photomicrographs were taken under a confocal laser-scanning microscope (Leica; Germany). Images shown are representative results of at least three biological replicates.
10
3D Coculture Model of CAFs and Macrophages
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Collagen type I (3 mg/mL; Solarbio, C8062), 1 mol/L NaOH solution (Acmec, S41251), 10× PBS (Servicebio, G0002) solution, and dH2O were mixed in a volume ratio of 40:1:6:13 to prepare a pH neutral collagen working solution. Aliquots of 300 μL were used to prepare three-dimensional (3D) collagen. After the primary CAF cells and macrophages were counted, 3.0 × 105 cells of each were mixed in equal proportions, dissolved in 3D collagen, and placed in 37°C and 5% CO2 atmosphere. After gelation, the 3D collagen system was covered with culture medium to construct a 3D coculture model. IF was used to detect the expression and localization of related indicators.
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