Paraffin sections were deparaffinized, hydrated in a graded ethanol series, and then boiled for 15 min in citrate antigen retrieval solution (Solarbio). The sections were then stained using a universal streptavidin-perosidase staining kit (Solarbio) as previously described [22 (link)]. Briefly, the sections were treated with 0.3% H2O2 for 30 min at 37 °C to quench endogenous peroxidase activity, washed with PBS, and blocked with pre-immune serum for 30 min at 37 °C. Following the manufacturer’s instructions, the sections were incubated with an anti-Snail (1:100, diluted in PBS, Santa Cruz Biotechnology, Inc., Dallas, Texas, USA, sc-393172) specific primary antibody at 4 °C. The secondary antibody was incubated at 37 °C for 1 h and then incubated with streptavidin–biotin peroxidase for 30 min at 25 °C. Finally, the sections were visualized with diaminobenzidine lightly counterstained with hematoxylin, dehydrated, and mounted with coverslips. Sections were viewed under a microscope (Nikon, Co., Tokyo, Japan) after drying at 25 °C.
Citrate antigen retrieval solution
Manufactured by Solarbio
Sourced in China
Citrate antigen retrieval solution is a laboratory reagent used to prepare tissue samples for immunohistochemical analysis. It is designed to enhance the exposure of target antigens, allowing for improved detection and visualization of the desired molecules in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Anti snail, by Santa Cruz Biotechnology (1 mentions)
Vimentin, by Merck Group (1 mentions)
Vimentin, by ZSGB-BIO (1 mentions)
Edta antigen retrieval solution, by ZSGB-BIO (1 mentions)
Eclipse e400, by Nikon (1 mentions)
Anti ki67 antibody, by Beyotime (1 mentions)
Dab kit, by ZSGB-BIO (1 mentions)
Ab214821, by Abcam (1 mentions)
Ab270993, by Abcam (1 mentions)
Ab228748, by Abcam (1 mentions)
A32731, by Thermo Fisher Scientific (1 mentions)
Fv1000 ix81 microscope, by Olympus (1 mentions)
Pshp2 y542, by Abcam (1 mentions)
α sma, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Confocal laser scanning microscope, by Leica (1 mentions)
Ab2601, by Abcam (1 mentions)
Gc305006, by Wuhan Servicebio Technology (1 mentions)
G0002, by Wuhan Servicebio Technology (1 mentions)
G1401, by Wuhan Servicebio Technology (1 mentions)
9 protocols using citrate antigen retrieval solution
1
Immunohistochemical Detection of Snail
2
Immunohistochemical Analysis of Vimentin and E-cadherin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunohistochemistry, heat-induced epitope retrieval was performed by Citrate Antigen Retrieval solution (#C1031, Beijing Solarbio Science & Technology Co., Ltd.) for 40 min for vimentin, or by EDTA Antigen Retrieval solution (#ZLI-9079, ZSGB-Bio) for E-cadherin. The tissue preparations were incubated with the primary antibodies for E-cadherin (#20874-1-AP, ProteinTech Group, Inc.,) at a 1:500 dilution, vimentin (#HPA001762, Sigma-Aldrich; Merck KGaA) at a 1:250 dilution at 4°C overnight, followed by incubation with the secondary antibody EnVision™+/HRP rabbit polymer (#P0448, Dako; Agilent Technologies, Inc.) at a 1:200 dilution at room temperature for 30 min. Secondary antibody detection was performed by using the SIGMAFAST™ 3,3′-diaminobenzidine tablets (DAB Peroxidase Substrate Tablet Set, #D4168, Sigma-Aldrich; Merck KGaA). The slides were counterstained with hematoxylin for 2 min following color separation by 1% acetic acid at room temperature for 30 sec. The Samples were visualized under a light microscope (Nikon, model Eclipse E400) and images were captured using a Nikon Digital Camera (ACT-1 Nikon software).
3
Immunohistochemical Analysis of MSX1 in Uterine Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Paraffin sections containing uterine tissue were deparaffinized and hydrated in graded ethanol series before staining with the streptavidin–peroxidase method. The antigens were retrieved by boiling for 15 min in citrate antigen retrieval solution (Solarbio, Beijing, China). Endogenous peroxidase was blocked by incubation in 3% hydrogen peroxide. Then, the instructions of the universal SP staining kit were followed (Maixin Biotechnologies, Fuzhou, China). Briefly, the sections were pretreated with 0.3% H2O2 for 40 min at 37 °C to quench the endogenous peroxidase activity; then, they were washed with phosphate-buffered saline (PBS). The sections were incubated with 10% serum for 60 min at 37 °C. After blocking, the sections were incubated with anti-MSX1 antibody (1:100, Abcam, ab168745, Cambridge, UK) overnight at 4 °C, and the second antibody was incubated at 37 °C for 2 h. The sections were then washed with PBS, then incubated with streptavidin-biotin peroxidase for 40 min at 25 °C. Thereafter, the sections were visualized with diaminobenzidine (DAB), lightly counterstained with hematoxylin for 25 s, dehydrated, and coverslipped. As a negative control, the primary antibody was substituted with pre-immune serum. The sections were imaged under a microscope (Nikon, Tokyo, Japan) after drying at 25 °C.
4
Immunohistochemical Analysis of Murine Pancreatic Tumors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IHC was conducted with reference to previous studies (Liao et al., 2019 (link); Wu et al., 2020 (link)). Paraffin sections of mouse pancreatic tumors were subjected to a standard xylene and alcohol gradient for deparaffination, and then antigen retrieval was carried out through Citrate Antigen Retrieval solution (Solarbio, China). Streptavidin-Biotin Assay Kit (ZSGB-BIO, China) was used for endogenous peroxidase blockade and antibody incubation. Sections were incubated with primary antibodies, including anti-Ki67 antibody (Beyotime, China) and anti-Ly-6G/Ly-6C (Gr-1) antibody (BioLegend, United States). After overnight at 4°C, sections were incubated with Biotin-conjugated Goat Anti-Rat or Rabbit IgG (Proteintech, United States). Sections were subsequently coloured by using DAB kit (ZSGB-BIO, China) and stained with hematoxylin. The expression of Gr-1 and Ki67 was quantitatively analyzed using ImageJ Fiji software according to the previous protocol (Crowe and Yue, 2019 (link)).
5
Immunohistochemical Analysis of Bone Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The paraffin sections of bone tissues were deparaffinized, rehydrated, and then incubated in the citrate antigen retrieval solution (Beijing Solarbio Science and Technology Co.,Ltd., C1031, China) for 5 min. After quenching endogenous peroxidase activity with 3% H2O2 for 8 min, the slides were incubated with anti-BMP-2 antibody (ab214821, Abcam, United States, 1:100), anti-COL1A1 antibody (ab270993, Abcam, United States, 1:100) and anti-OPN antibody (ab228748, Abcam, United States, 1:100) at 4°C overnight. On the next day, the slides were incubated at 37°C for 20 min with goat anti-rabbit IgG (A32731, Invitrogen, United States, 1:500). After 3,3′-diaminobenzidine (DAB) (Gene Tech Company Ltd., GK5007, China) staining, the slides were counterstained with hematoxylin for 3 min at room temperature, dehydrated in a series of 70–100% alcohol baths and cleared in a xylene bath. The slides were mounted with neutral balsam and observed using a biological microscope (Olympus, BX53, Japan).
6
Immunohistochemical Analysis of Lung Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lung sections were incubated at 65°C for 1 h, then heated in citrate antigen-retrieval solution for 30 min (Solarbio). 0.5% TritonX-100 was used for permeabilization and 5% FBS was used for blocking. Sections were incubated with primary antibodies against Jag1 (Cell Signaling Technology), Shp2 (Sigma-Aldrich), pShp2 Y542 (Abcam), IB4 (Invitrogen), α-SMA (Cell Signaling Technology), Arg1 (Proteintech Group), and CD68 (Ebioscience) overnight at 4°C. The matched fluorescein-linked secondary antibodies (Invitrogen) were used for visualization by incubation at room temperature for 1 h. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI, Beyotime). Images were acquired with an Olympus IX81-FV1000 microscope.
Cells on slides were fixed with 4% paraformaldehyde for 20 min, then permeabilized with 0.1% TritonX-100 for 20 min.
Cells on slides were fixed with 4% paraformaldehyde for 20 min, then permeabilized with 0.1% TritonX-100 for 20 min.
7
Immunofluorescent Detection of UCP1 in Paraffinized Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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.
8
Immunohistochemical Analysis of Tumor Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Paraffin-embedded sections prepared from the in vivo experiments were used for immunohistochemistry assays to detect the protein expression levels of Ki-67 and BCL2. Briefly, the sections were deparaffinized, rehydrated, submerged in citrate antigen retrieval solution (Solarbio, China) for heat-induced antigenic retrieval, immersed in 0.3% hydrogen peroxide to block endogenous peroxidase activity, blocked with 5% goat serum, incubated with primary antibody at 4 °C overnight and developed using a DAKO ChemMate Envision Kit/HRP (Dako-Cytomation, USA). Then, the sections were counterstained with hematoxylin, dehydrated, cleared and mounted with neutral gums. Protein expression was determined by randomly selecting three tumor cell areas for each specimen under the same conditions using Image-pro plus 6.0 software. The mean optical density (MOD) was calculated with the following formula: MOD = IOD SUM / area SUM (IOD: the integrated optical density; IOD SUM: the cumulative IOD of the targeted areas in one photo; area SUM: the sum of the targeted areas). In addition, PBS was substituted for the primary antibody for the blank control. See Additional file 1 : Table S1 for the antibodies used.
9
Immunohistochemical Analysis of S100A7
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Para n sections were depara nized and hydrated in graded ethanol series before staining with the streptavidin-perosidase method. Antigens were retrieved by boiling for 15 min in Citrate Antigen Retrieval solution (Solarbio, China). Endogenous peroxidase was blocked by incubation in 3% hydrogen peroxide.
Then followed the instructions of the universal SP staining kit, in which the primary antibody anti-S100A7 (1:100, Bioss, Beijing, China) is incubated overnight at 4°C, and the second antibody is incubated at 37°C
for 1 hour. After DAB incubation, sections were lightly counterstained with hematoxylin and were dehydrated and cover slipped.
Then followed the instructions of the universal SP staining kit, in which the primary antibody anti-S100A7 (1:100, Bioss, Beijing, China) is incubated overnight at 4°C, and the second antibody is incubated at 37°C
for 1 hour. After DAB incubation, sections were lightly counterstained with hematoxylin and were dehydrated and cover slipped.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!