Paraffin-embedded sections of formalin-fixed lymph node biopsies (5 μm) were blocked using 0.3% H2O2 and placed on APES-coated slides. Antigens were cooked for 3 min in citrate buffer (Solarbio Life Sciences, Beijing, China) to achieve retrieval. Mouse monoclonal anti-human CD42a antibody (ThermoFisher, Waltham, Massachusetts, USA) and rabbit monoclonal anti-human CD4 antibody (Zhongshan Goldenbridge Biotech, Beijing, China) were added to the slides at 4°C overnight. 4’,6-diamidino-2-phenylindole (DAPI, blue color) was used to stain the nuclei as previously described (36 (link)). Images were obtained using a Leica Biosystems Aperio VERSA scanning system (Leica, Richmond, Virginia, USA).
Citrate buffer
Manufactured by Solarbio
Sourced in China
Citrate buffer is a common laboratory solution used to maintain a specific pH level in various biochemical experiments and applications. It is prepared by mixing citric acid and sodium citrate in appropriate proportions to achieve the desired pH value. The citrate buffer helps to create a stable and controlled environment for reactions and analyses, ensuring consistent and reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
3 3 diaminobenzidine (dab), by Solarbio (2 mentions)
Axio observer 3, by Zeiss (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Beyotime (1 mentions)
Toluidine blue, by Solarbio (1 mentions)
Ar1109, by Boster Bio (1 mentions)
Sirius red staining, by Solarbio (1 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
Axio vert a1, by Zeiss (1 mentions)
Fitc conjugated secondary antibody, by Beyotime (1 mentions)
Anti jak1 phospho tyr1022, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Fluorescent microscopy, by Olympus (1 mentions)
F4 80, by Cell Signaling Technology (1 mentions)
Strept avidin biotin complex, by Boster Bio (1 mentions)
Abc staining system, by Bioss Antibodies (1 mentions)
Optical microscope, by Leica (1 mentions)
Antibody against f4 80, by Abcam (1 mentions)
Goat serum, by Solarbio (1 mentions)
Hematoxylin, by Solarbio (1 mentions)
16 protocols using citrate buffer
1
Immunohistochemistry of Lymph Node Biopsies
2
Immunohistochemical Analysis of IL-6 in Lung Tissue
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Lung and heart tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and then sliced into sections 5 μm thick. Lung tissue sections were stained with hematoxylin and eosin (HE) according to standard protocols. For immunohistochemical staining, the tissue sections were baked at 65°C for 2 h, deparaffinized and rehydrated, performed to antigen retrieval in 0.01 M citrate buffer (Solarbio biotechnology Beijing, China). Then, tumor tissues were blocked with 5% goat serum for 1 h and then incubated with IL-6 antibody (1:200, santa, USA) at 4°C overnight. After washing, HRP-secondary antibody was added at room temperature for 1 h. At last, antibody slides were counterstained with hematoxylin. The image was captured through microscope (Nikon, Shinagawa, Tokyo, Japan).
3
Histological Analysis of Collagen XVII and Integrin β4
4
Immunofluorescence Analysis of Pancreatic Tissue
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Following fixation with 4% paraformaldehyde, mouse pancreatic tissue sections were processed for embedding in paraffin, and then deparaffinized utilizing xylene and ethanol. The sections were treated with Citrate buffer (C1010, Solarbio, Beijing, China) which was heated and subsequently immersed. The mixture was then reduced to low heat and left to process for a duration of 15 min. To prevent nonspecific binding, the sections were blocked with a solution containing 3% hydrogen peroxide for a duration of 20 min. Sections were incubated with either insulin or glucagon primary antibodies overnight at 4 °C, then incubated with secondary antibodies for 1 h. Cell nuclei were stained with a DAPI solution, and sections were blocked with an anti-fluorescence quenching sealer (AR1109, Boster, Wuhan, China). Lastly, the sections were captured using an inverted fluorescence microscope (NIB900, Leica Microsystems, Germany).
5
Histological Analysis of Heart Tissue
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The heart tissue was fixed with 4% formaldehyde, followed by dehydrated, transparent, and embedded with paraffin. Finally, the heart tissue was cut into 5 μm thick samples for subsequent experiments. For immunohistochemical staining, the tissue sections were baked at 65 °C for 2 h, deparaffinized and rehydrated, and then performed to antigen retrieval in 0.01 M citrate buffer (Solarbio Biotechnology Co., Ltd, Beijing, China). Then, tissues were blocked with 5% goat serum for 1 h and then incubated with VEGF-B antibody (1:200) at 4 °C overnight. Afterwards, the slices were stained with hematoxylin and eosin (H&E) (Beyotime Biotechnology, Shanghai, China), Masson staining (ZSGB-BIO, Beijing, China), WGA-FITC staining (Sigma-Aldrich, St. Louis, MO, USA), and Sirius red staining (Solarbio Biotechnology Co., Ltd, Beijing, China) according to the manufacturer’s instructions. In the histological staining described above, the magnifications for H&E, Masson, and Sirius Red were 200x, the magnification for WGA staining was 400x. The image was captured under microscope (Nikon, Tokyo, Japan).
6
Immunofluorescence Assay for Tissue Characterization
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Immunofluorescence (IF) assay were performed on the frozen sections according to the previously established procedures.33 Briefly, sections were treated with pepsinum (ZSGB Biotechnology, Beijing, China) at 37°C for 15 minutes or 0.01 mol/L citrate buffer (Solarbio, Beijing, China) at 60°C for 4 hours. Next, sections were incubated in primary antibodies overnight at 4°C and the antibodies applied in this study included TGF‐β1 (diluted 1:200, Arigo Biolaboratories, Shanghai, China), Phospho‐Smad2 (p‐Smad2; diluted 1:200, Thermo Fisher Scientific, Pittsburgh, PA, USA), type II collagen (Col‐II; diluted 1:200, Abcam, Cambridge, UK), osteocalcin (OCN; diluted 1:200, Takara, UK) and CidU (diluted 1:100, Abcam, Cambridge, UK). After incubation with secondary antibodies for 20 minutes, tissue sections were counter‐stained with 4',6‐diamidino‐2‐phenylindole (DAPI). Fluorescent quantitative analysis was calculated from three mice (one representative section per mouse) using image‐pro plus software.
7
Quantifying PDCoV Viral Attachment and Internalization
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For the viral attachment assays, LLC-PK cells treated with HSP90 inhibitors, 17-AAG and VER-82576, or HSP90AB1WT and HSP90AB1KO cells were incubated with PDCoV (MOI = 10) for 1 h at 4 °C. Unattached virus was removed by washing three times with cold PBS. Cells were then collected to quantify the amount of bound virus, and the level of PDCoV M mRNA was determined by qRT–PCR. For the viral internalization assay, cells were incubated with PDCoV (MOI = 10) for 1 h at 4 °C to allow for viral attachment. Infected cells were washed three times with cold PBS and incubated at 37 °C for another 1 h to allow for viral internalization. Cells were then washed three times with cold citrate buffer (pH = 3) (Solarbio) to remove the bound, but noninternalized, viral particles. The cells were collected, and the level of PDCoV M mRNA was determined by qRT–PCR.
8
Immunohistochemistry of NICD Protein
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Paired GC and normal mucosal specimens were embedded in paraffin and cut into 4-µm-thick sections and mounted on glass slides. Following mounting, they were kept in an oven at 70°C. Sections were then deparaffinized with xylene twice for 5 min and rehydrated with ethanol gradient (100, 95, 90, 80 and 70%, diluted in double distilled water) for 2 min at room temperature. The sections were blocked with 1% H2O2 at room temperature for 30 min against the endogenous peroxidase activity, treated for antigen retrieval by microwave in Citrate buffer (10 mmol/l, pH 6.0, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for 10 min, washed with PBS and incubated with goat serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) blocking solution for 1 h at room temperature. The blocked sections were incubated with the primary antibodies against NICD (cat. no. 3608; Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4°C at a dilution of 1:50 and then incubated with biotin-labeled anti-rabbit IgG (1:1,000; cat. no. ab6720; Abcam, Cambridge, MA, USA) for 1 h at room temperature. A total of 10 randomly selected fields under ×400 magnification were observed under a light microscope (Carl Zeiss AG, Oberkochen, Germany).
9
Quantitative Histological Analysis of Femur Vascularity
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After μCT scanning, the femur samples were processed for paraffin sections at the thick of 3 μm as previously described [21 (link)]. Alcian Blue Hematoxylin/Orange G (ABH) staining was performed on these sections for morphological analysis. The numbers of blood vessel and trabecular area (%) in the region of interest were measured using OsteoMetrics software (Decatur, GA, USA) by two researchers. The IHC assay was detected as follows: (1) Sections were treated with 0.01 M citrate buffer (Solarbio, Beijing, CN) at 60 °C for 4 h as antigen retrieval; (2) sections then were incubated in primary antibodies of CD31 (Diagbio, AGR52748, CN), cyclooxygenase-2, (COX2, Huanbio, RT1159, CN), endothelial nitric oxide synthase (eNOS, Huanbio, R1412-3, CN) and vascular endothelial growth factor (VEGF, ARIGO, ARG10513, CN) overnight at 4 °C; (3) sections were incubated in secondary antibodies for 20 min and diaminobenzidine (DAB) solution for 1 min to detect positive staining; and (4) sections were counterstained with hematoxylin. ImageJ software was used to analyze the quantification of positive staining.
10
Immunofluorescence Staining of Paraffin Sections
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Four-micrometer paraffin sections were baked, deparaffinized, and rehydrated in xylene and graded alcohols; antigen retrieval was then performed using citrate buffer (Cat# C1032, Solarbio, Beijing, China) for 20 min at 100 °C within a pressure chamber. Slides were blocked using normal 10% goat serum or donkey serum for 1 h at room temperature, and subsequently incubated with indicated primary antibodies (Dilution: 1:200) at 4 °C overnight. Sections were then incubated for 1 h at 37 °C with the fluorescein-conjugated secondary antibodies, and the sections were then counterstained with DAPI. The negative control consisted of sections processed in the same way as the tests with the omission of the primary antibody incubation step. Images were examined through Carl Zeiss (Axio Vert.A1) fluorescence microscopy. All experiments were performed three times. There were four mice in each experimental group, and two sections per mouse were randomly selected for imaging. Three fields of the image were randomly selected for each section for counting. Counting was performed in a blinded manner. Data analysis was performed using ImageJ software. All antibodies used in the study are shown in Table 1 .
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