Liver tissues were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) overnight and sliced into 5-μm-thick paraffin-embedded sections. After deparaffinization and rehydration, antigen retrieval was performed by boiling the sections in citrate antigen retrieval solution (P0081; Beyotime) for 15 min. Next, the endogenous peroxidase activity of the samples was blocked by 3% H2O2 for 15 min, followed by pre-blocking with 5% BSA (A1933; Sigma–Aldrich) for 1 h at room temperature. The levels of proteins were investigated in liver tissues using primary antibodies against BMAL1 (Abcam, 1:800), FVII (Santa Cruz Biotechnology Inc., 1:400), and FXII (Santa Cruz Biotechnology Inc., 1:400) using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA), followed by the DAB Substrate kit (Vector Laboratories). All protocols were followed according to the manufacturer's instructions.
Citrate antigen retrieval solution
Manufactured by Beyotime
Sourced in China
Citrate antigen retrieval solution is a laboratory reagent used to expose antigenic sites within biological samples, such as tissue sections, for immunohistochemical (IHC) analysis. It is a buffer solution that helps to unmask the target antigens, enabling better detection and visualization during IHC procedures.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Beyotime (3 mentions)
Immpact dab, by Vector Laboratories (2 mentions)
Vectastain elite abc kit, by Vector Laboratories (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Dab horseradish peroxidase color development kit, by Beyotime (2 mentions)
Bmal1, by Abcam (1 mentions)
A1933, by Merck Group (1 mentions)
Dab substrate kit, by Vector Laboratories (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
Neuronal nuclei (neun), by Cell Signaling Technology (1 mentions)
Anti bax antibody, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Smi 31, by Abcam (1 mentions)
Luxol fast blue stain, by Abcam (1 mentions)
Sp 9002, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Zli 9018, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Goat anti rabbit igg alexa fluor plus 647, by Thermo Fisher Scientific (1 mentions)
Confocal laser scanning microscope, by Zeiss (1 mentions)
Rabbit anti f4 80, by Wuhan Servicebio Technology (1 mentions)
Glp 1r, by Santa Cruz Biotechnology (1 mentions)
36 protocols using citrate antigen retrieval solution
1
Immunohistochemical Analysis of Liver Proteins
2
Quantifying Neuronal Apoptosis via TUNEL
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A TUNEL (terminal deoxynucleotidal transferase–mediated biotin–deoxyuridine triphosphate nick‐end labeling) staining kit (11684817910, Roche Holding AG, Basel, Switzerland) was used according to the manufacturer’s instructions. Briefly, brain slices were fixed in 4% paraformaldehyde for 15 minutes, washed 3 times in PBS, and then antigen retrieval was performed using Citrate Antigen Retrieval Solution (P0081, Beyotime Biotechnology, Shanghai, China). After permeabilizing and blocking by 5%‐bovine serum albumin‐0.25% Triton‐X100 in PBS for 30 minutes at room temperature, slices were incubated with a mixture of Enzyme Solution and Label Solution (1:9) for 1 hour at 37 °C. Slices were incubated with primary antibody NeuN (24307, Cell Signaling Technology, Inc.) overnight at 4 ℃ after washing 3 times in PBS. The remaining steps were the same as in immunofluorescent staining.
3
Immunohistochemical Analysis of Tissue Samples
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Harvested samples were fixed in 10% formalin for 24 h, followed by decalcification in 10% EDTA (pH 8) for 7 days and embedding in paraffin. Samples were then cut into 4-µm-thick sections for hematoxylin and eosin (HE) and immunohistochemical staining. After deparaffinization and rehydration, sections underwent antigen retrieval with citrate antigen retrieval solution (Beyotime, Shanghai, China) at 100°C for 5 min, blocking with 5% goat serum for 1 h at room temperature, and then incubated with primary antibodies (anti-MMR, Abcam, 1:200; and anti-iNOS, Santa, 1:200) overnight at 4°C. The next day, sections were washed with PBS and incubated with secondary antibodies (Yeason, Shanghai, China) for 1 h at room temperature. Nuclei were stained with DAPI, and the sections were washed and then visualized via laser scanning confocal microscopy.
4
Immunohistochemical Analysis of nNOS Expression
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Polygonati Rhizoma (Anhui Wansheng Chinese Herbal Slices Co., Ltd.), Achyranthis Bidentatae Radix (Shanghai Qingpu Chinese Medicine Tablet Co., Ltd.), Epimedii Folium (Hebei Grain Pharmaceutical Co., Ltd.) were purchased from Shanghai Bailutang Traditional Chinese Medicine Store (Shanghai, China).
Tissue Fixative Solution (G1101-500 ML), Phosphate Buffered Saline (G4202-500 ML), and Phosphate Buffered Saline Powder (G00002-2 L) were purchased from Wuhan Servicebio Technology Co., Ltd. (Wuhan, China). Ethanol absolute (10009218) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Xylene (X820585-500 ml) and Acetic acid (A801295-500 ml) were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). TritonX-100 (ZLI-9308), Rabbit SP Detection Kits (Biotin-Streptavidin HRP Detection Systems) (SP-9001), DAB Kits (ZLI-9018) and Hematoxylin Staining Solution (ZLI-9610) were purchased from Beijing Zhong Shan-Golden Bridge Biological Technology Co., Ltd. (Beijing, China). Citrate Antigen Retrieval Solution was purchased from Beyotime Biotech Inc. (Shanghai, China). The nNOS (C7D7) Rabbit mAb (#4231S) were purchased from Cell Signaling Technology, Inc (Boston, US).
Tissue Fixative Solution (G1101-500 ML), Phosphate Buffered Saline (G4202-500 ML), and Phosphate Buffered Saline Powder (G00002-2 L) were purchased from Wuhan Servicebio Technology Co., Ltd. (Wuhan, China). Ethanol absolute (10009218) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Xylene (X820585-500 ml) and Acetic acid (A801295-500 ml) were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). TritonX-100 (ZLI-9308), Rabbit SP Detection Kits (Biotin-Streptavidin HRP Detection Systems) (SP-9001), DAB Kits (ZLI-9018) and Hematoxylin Staining Solution (ZLI-9610) were purchased from Beijing Zhong Shan-Golden Bridge Biological Technology Co., Ltd. (Beijing, China). Citrate Antigen Retrieval Solution was purchased from Beyotime Biotech Inc. (Shanghai, China). The nNOS (C7D7) Rabbit mAb (#4231S) were purchased from Cell Signaling Technology, Inc (Boston, US).
5
Bax Protein Detection in Paraffin Sections
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Paraffin sections were deparaffinized and rehydrated. Antigen retrieval was performed according to the manufacturer's instructions for the Citrate Antigen Retrieval Solution (Beyotime, China). Sections were incubated in hydrogen peroxide to quench any endogenous peroxidases and then blocked with 5% bovine serum albumin (Sigma) for 30 min at room temperature. Sections were incubated in primary rabbit anti-Bax antibodies (1 : 100; Abcam) diluted in PBS overnight at 4°C. The Vectastain Elite ABC Kit (Vector Laboratories, USA) was used according to the manufacturer's instructions. Positive staining was visualized with DAB (ImmPACT DAB, Vector Laboratories, USA). Sections were counterstained with hematoxylin for 10 s and dipped in acid alcohol as needed before being dehydrated and mounted on coverslips.
6
Myelin and Axon Immunohistochemical Staining
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Sections were stained for myelin with the use of luxol fast blue stain (LFB; Abcam) and for axons using immunohistochemical staining for phosphorylated neurofilaments (SMI-31; Abcam; Budde et al., 2007 (link)). Immunohistochemical staining for SMI-31 was performed as described previously (Budde et al., 2007 (link)). After accepting antigen retrieved with the use of citrate antigen retrieval solution (P0081; Beyotime), the tissue sections were treated with 3% hydrogen peroxide to inactivate endogenous peroxidase. The sections were then incubated in 1:20 goat serum for 30 min, rinsed, and incubated overnight at 4°C with a 1:200 dilution of the primary antibody SMI-31. After three washes in PBS, the sections were incubated for 90 min with biotinylated goat anti-mice immunoglobulin G antibody (SP-9002; Zhongshan Golden Bridge Biotechnology). After another three PBS washes, the brain sections were incubated with avidin biotinylated horseradish peroxidase (SP-9002; Zhongshan Golden Bridge Biotechnology) for 90 min. The brain sections were rewashed three times in PBS and then incubated with diaminobenzidine and hydrogen peroxide (ZLI-9018; Zhongshan Golden Bridge Biotechnology). The nucleus was stained with hematoxylin. The sections were then rinsed in water for 10 min, dehydrated, and covered with a coverslip for microphotography.
7
Immunofluorescence Staining of Piglet Liver
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Liver segments of piglets were subjected to immunofluorescence staining. Briefly, six micron-thick slices were obtained from paraffin-embedded livers. Xylene and a series of alcohols were used for section dewaxing. After being soaked in citrate antigen retrieval solution (Beyotime Biotechnology, P0081, Shanghai, China), 0.3% Triton X-100 (Beyotime Biotechnology, ST797), and 3% H2O2-methanol, slices were blocked with 5% BSA for 1–2 h at room temperature. Next, specimens were incubated at 4 °C overnight with rabbit-anti-F4/80 (Servicebio, GB113373, Gent, Belgium) and cultured for 1 h with the secondary antibody, Alexa Fluor Plus 647 goat anti-rabbit IgG (Invitrogen, A32733). After all antigens had been labeled, nuclei were stained with Hoechst 33342 (Invitrogen, H3570). Images were acquired using a confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany) and analyzed by ImageJ program V1.8.0.112 (National Institutes of Health, Bethesda, MD, USA).
8
Immunofluorescence Analysis of GLP-1R and PPAR-γ in Rat Brain
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Fresh brains of rats were isolated and embedded by OTC (Sakura Finetek Japan Co., Ltd, Tokyo, Japan), and then, they were cut to get frozen 10μm sections. After slices were repaired in the high-pressure cooker by citrate antigen retrieval solution (Beyotime, Shanghhai, China, Catalogue No.: P0081) and blocked by 5% normal goat serum (Beyotime, Shanghai, China, Catalogue No.: C0265) (1 hour at room temperature). They were incubated with rabbit-anti-rat primary antibody to GLP-1R (Santa Cruz Biotechnology, Inc., Dallas, TX, USA, Catalogue No.: sc-390774) and PPAR-γ (Santa Cruz Biotechnology, Inc., Dallas, TX, USA, Catalogue No.: sc-390740) at 4° C overnight. After washing for 3 times, Alexa Fluor 594 conjugated goat-anti-rabbit secondary antibody (Proteintech, Wuhan, China, Catalogue No.: SA00013-3) was used to detect the primary antibody. Finally, sections were stained by DAPI (Beyotime, Shanghhai, China, Catalogue No.: P0131) and washed for 3 times with PBS. GLP-1R (PPAR-γ) and DAPI (in hippocampal dentate gyrus and cortex) were observed by a fluorescence microscope (Olympus, Japan) after triggered at 594 nm and 358 nm respectively. Image were captured by (CellSens Standard).
9
Immunohistochemical Analysis of Paraffin-Embedded Tissues
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Paraffin‐embedded were fixed with 4% paraformaldehyde overnight at room temperature and embedded in a paraffin block. Paraffin‐embedded slides were deparaffinized and rehydrated in a series of ethanol solutions. After two washes with PBS for 5 minutes each, antigen retrieval was performed in Citrate Antigen Retrieval Solution (Beyotime) by boiling for 10 minutes. After cooling down, slides were blocked with 10% foetal bovine serum in PBS for 1 hours. Then, various primary antibodies (Ki67, C CASP3 and E‐cadherin) were applied in a concentration of 8 μg/mL overnight at 4°C. After washed with PBS, HRP‐conjugated secondary antibodies were added on the slides for incubating 1 hour. DAB substrate solution was used to reveal the colour of antibody staining. The intensity was scored as follows: 0, none; 1, weak; 2, moderate; and 3, intense.
10
Immunohistochemical Detection of Cleaved Caspase-3
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Tissue sections were deparaffinized and rehydrated with xylene and ethanol, as aforementioned. Subsequently, the antigen was retrieved using citrate antigen retrieval solution (Beyotime Institute of Biotechnology) and blocked with 5% goat serum (AmyJet Scientific, Inc.) diluted in TBS + 0.5% Tween 20 (TBST), the sections were probed with the primary antibody against cleaved caspase3 (c-caspase3; 1:100; cat. no. 9661; Cell Signaling Technology, Inc.) at 4°C overnight. Following the primary antibody incubation, sections were incubated with a horseradish peroxidase-conjugated secondary antibody (1:500; cat. no. 18653; Cell Signaling Technology, Inc.) at room temperature for 1 h, followed by incubation with the chromogen 3,30-diaminobenzidine tetrachloride (R&D Systems, Inc.) for 2–3 sec at room temperature. Cell nuclei were stained with 1% Harris hematoxylin solution for 30 sec at room temperature. The staining of c-caspase3 was evaluated by two pathologists on the basis of the positive staining proportion and the staining intensity (24 (link)). The positive staining percentage was scored as 0 for ≤5%, 1 for 6–25%, 2 for 26–50%, 3 for 51–75% and 4 for ≥75%, respectively. Intensity was marked as follows: 0, no staining; 1 weak staining; 2, moderate staining; and 3, strong staining. The final score was obtained by multiplying the percentage score and intensity score.
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