Cells in chamber slides were washed with 1× PBS (Lonza) and fixed in 4% paraformaldehyde for 15 min followed by permeabilisation in blocking buffer (1× PBS/1% BSA) containing 0.3% Triton X-100 for 1 h at room temperature and incubation with primary and secondary antibodies diluted in 1× PBS/1% BSA/0.3% Triton X-100 for 12–16 h at 4 °C and 60 min at room temperature, respectively. Nuclei were counterstained with DAPI (2 μg ml−1) (Sigma) for 3 min at room temperature. For the staining of paraffin-embedded tissue, 2–3 μm sections were heated to 60 °C for 30 min, followed by deparaffinisation and rehydration in xylene and descending ethanol solutions. Heat-mediated antigen retrieval was performed by boiling in citrate buffer (SignalStain Citrate Unmasking Solution, Cell Signaling). After permeabilisation in 1× PBS/0.1% Triton X-100, sections were blocked in 1× PBS/1% BSA/0.3% Triton X-100 for 1 h at room temperature. Antibody incubation and counterstaining with DAPI was performed as described for the staining of tissue culture cells. Images were taken on a Leica TCS SP8 confocal microscope. For the quantification of Cyclophilin A-positive, murine cells in teratoma tissue, five to eight images per sample were counted using Photoshop. The amount of murine cells was evaluated in relation to 100 teratoma cells.
Signalstain citrate unmasking solution
Manufactured by Cell Signaling Technology
Sourced in United States
SignalStain Citrate Unmasking Solution is a laboratory reagent used for antigen retrieval in immunohistochemistry (IHC) and other related techniques. The solution is designed to unmask or expose antigenic sites that may be hidden or obscured within formalin-fixed, paraffin-embedded (FFPE) tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Signalstain antibody diluent, by Cell Signaling Technology (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Lonza (1 mentions)
Tcs sp8 confocal microscope, by Leica (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Alexa fluor 555 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 conjugate, by Thermo Fisher Scientific (1 mentions)
Bmk 2202, by Vector Laboratories (1 mentions)
Protein block, by Agilent Technologies (1 mentions)
Rabbit anti p erk, by Cell Signaling Technology (1 mentions)
Cst 2965, by Cell Signaling Technology (1 mentions)
Signalstain boost detection reagent, by Cell Signaling Technology (1 mentions)
Cst9101, by Cell Signaling Technology (1 mentions)
Sc 365106, by Santa Cruz Biotechnology (1 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Diaminobenzidine staining kit, by ZSGB-BIO (1 mentions)
Mounting medium, by Cell Signaling Technology (1 mentions)
Ventana benchmark special stain platform, by Roche (1 mentions)
Anti ki 67 d3b5, by Cell Signaling Technology (1 mentions)
Hrp conjugated secondary antibody, by Vector Laboratories (1 mentions)
7 protocols using signalstain citrate unmasking solution
1
Immunofluorescence Staining of Cells and Tissues
2
Immunofluorescent Skeletal Muscle and Lung Analysis
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Skeletal muscles and lungs were fixed for 16 hours at 4°C in 4% paraformaldehyde, and routine paraffin embedding and H&E staining were performed. For immunofluorescence, antigen retrieval was performed with SignalStain Citrate Unmasking Solution (Cell Signaling Technology, 14746) per the provider’s instructions. Sections were then permeabilized in 0.3% Triton X-100 for 15 minutes and blocked with mouse-on-mouse blocking solution (Vector Laboratories, BMK-2202) and 5% goat serum. The following antibodies were used at 1:200 dilution: ACTA1 (Proteintech, 17521-1-AP), wheat germ agglutinin and Alexa Fluor 647 conjugate (Thermo Fisher Scientific, W32466), and anti-rabbit Alexa Fluor 555 secondary antibody (Thermo Fisher Scientific, A32732). A Zeiss LSM 800 was used for image acquisition.
3
Immunohistochemical Analysis of B7x Expression
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Prior to staining, slides were baked at 60°C overnight. FFPE sections were de-paraffinized and hydrated by washing in xylene and decreasing concentrations of ethanol (100%, 95%). Antigen retrieval was performed by first boiling the samples in signal stain citrate unmasking solution (Cell signaling technology) for 2 mins. Then, the slides were placed in a steamer for additional 20 minutes. Slides were incubated in 3% hydrogen peroxide for 10 mins to block endogenous peroxidase. Non-specific binding of IgG was blocked using serum-free Protein block (Dako). Sections were labeled with primary antibody anti- human B7x (clone D1M8I, Cell signaling technology) overnight at 4°C. Secondary HRP conjugated goat anti- rabbit (Cell signaling technology) was added subsequently, followed by addition of chromogen DAB (Cell signaling technology) and hematoxylin nuclear counterstaining. Images were acquired using Zeiss Axio Observer CLEM microscope. The intensity of the immunoreactivity was scored as 0 (none), 1 (low), 2 (medium), or 3 (high).
4
Immunohistochemical Analysis of FGF2 and pAkt/pERK
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Freshly harvested tissues were fixed in 4% paraformaldehyde (PFA) overnight, and IHC was performed using a SignalStain Citrate unmasking solution (Cell Signaling, 14746) and SignalStain Boost detection reagent (Cell Signaling, 8114) according to the manufacturer’s instructions. Primary antibodies used were rabbit anti-Fgf2 (SC 365106, 1:200; Santa Cruz Biotechnology), rabbit anti-pAkt308 (CST2965, 1:250; Cell Signaling), and rabbit anti–p-Erk (CST9101, 1:100; Cell Signaling).
5
Immunohistostaining of Ki67 in FFPE Tumor Tissue
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Immunohistostaining of Ki67 was performed on 4-mm sections of formalin-fixed paraffin-embedded (FFPE) tumor tissue using VENTANA BenchMark Special Stain platform (Roche, Indianapolis, IN, USA). Briefly, the section was dehydrated and antigen unmasking was performed by heating the section submersed in 1X citrate unmasking solution (SignalStain® Citrate Unmasking Solution, Cell Signaling Technologies, #14,746) for 10 min at a sub-boiling temperature (95°-98°C). After cooling, sections were washed in dH2O three times and then incubated in 3% hydrogen peroxide for 10 min. The section was blocked 1 h at room temperature in TBST buffer with 5% Normal Goat Serum, and then incubated with primary antibody anti-Ki-67 (D3B5) (Cell Signaling Technologies, #9129) diluted 1:500 in SignalStain® Antibody Diluent (#8112) overnight at 4°C. After 3 washes with TBST buffer, the section was incubated with HRP-conjugated secondary antibody (1:3000 dilution; Cell Signaling Technologies, #7074) for 60 min at room temperature. After wash, the section was stained by a diaminobenzidine staining kit (ZSGB-BIO, Beijing, China) for 30 min at room temperature. The section was washed in dH2O two times and then dehydrated. Section was mounted with coverslips using the mounting medium (Cell Signaling Technologies, #14,177) before imaging.
6
Immunohistochemical Detection of Myc in FFPE Tissues
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Slides with FFPE sections were warmed for 20 to 30 minutes in a 60°C oven before deparaffinization. The sections were then deparaffinized with xylene 3 × 5 minutes, rehydrated by incubation in 100% ethanol 2 × 5 minutes, 90% ethanol 1 × 3 minutes, 70% ethanol 2 × 3 minutes, 50% ethanol 1 × 3 minutes, 30% ethanol 1 × 3 minutes, and 2 × 10 minutes in deionized water and stained with hematoxylin and eosin (H&E). Post rehydration, antigen retrieval was performed in SignalStain Citrate Unmasking Solution (Cell Signaling Technology, cat no. 14746S) at 98°C for 10 to 20 minutes. Following antigen retrieval and inhibition of endogenous peroxidase activity with 3% H2O2 for 15 minutes, the slides were incubated with 10% normal goat serum for 1 hour at room temperature. Tissue sections were incubated with primary antibody: Myc (1:100; Cell Signaling Technology, 5605S) diluted in SignalStain Antibody Diluent (Cell Signaling Technology, cat no. 8112) overnight at 4°C in a humid chamber. Following 3 × 5-minute washes in TBST, tissue sections were incubated with HRP-conjugated secondary antibody (Vector Laboratories) for 1 hour at room temperature followed by development using chromogenic substrate DAB (SignalStain DAB Substrate Kit, Cell Signaling Technology, cat no. 8059s) for 10 minutes at room temperature. Slides were scanned and images were captured using a NanoZoomer (Hamamatsu).
7
Immunohistochemical Staining for CD68
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Tissue slides for immunochemistry staining were prepared as described above. For CD68 IHC staining, sections were pre-treated using heat-mediated antigen retrieval with SignalStain Citrate Unmasking Solution (Cell Signaling Technology, USA), followed by washing with Dulbecco’s Phosphate Buffered Saline (DPBS, Solarbio). Blocking of endogenous peroxidases was carried out with treatment in a solution of 30% methanol, 3% H2O2 in PBS for 30 min, and blocking of nonspecific binding was performed by incubation in PBS supplemented with 5% goat serum, 1% BSA and 0.3% Triton 100X (Sigma-Aldrich). Sections were then incubated with rabbit anti-CD68 antibody (1:100, Abcam, Cambridge, MA, USA) at 4°C overnight. The enhanced goat anti-rabbit biotinylated secondary antibody (ZSGB-BIO, Beijing, China) was used to detect the primary antibody and visualized using an HRP conjugated ABC system (ZSGB-BIO). DAB was used as the chromogen. Sections were then counterstained with hematoxylin, dehydrated through ethanol, cleaned in Histo-Clear, and mounted with neutral balsam. All images were taken using an optical microscope (Nikon ECLIPSE CI-S) with an image analysis system (Nikon DS-U3).
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