AP activity was measured using an Alkaline Phosphatase Detection Kit (System Biosciences) according to the manufacturer's instructions. For immunocytochemistry, NMR-iPSCs and differentiated cells were fixed with 4% paraformaldehyde in PBS for 5 min at room temperature, washed with PBS and then treated with 0.3% Triton X-100 in Tris-NaCl-blocking buffer (PerkinElmer) for 60 min at room temperature. The cells were incubated with primary antibodies against Oct4 (Millipore; 7F9.2; 1:200) and E-cadherin (BD Pharmingen; clone 36; 1:500) for 12 h at 4 °C. After washes with PBS, the cells were incubated with secondary antibody Alexa Fluor 555 anti-Ms IgG (Cell Signaling Technology (CST); A21424; 1:1,000) and Alexa Fluor 555 anti-rabbit IgG (CST; A21429; 1:1,000), and nuclei were counterstained with 1 μg ml−1 Hoechst 33,258 (Sigma Aldrich) for 60 min at room temperature. After washes with PBS, the images were captured.
Tris nacl blocking buffer
Manufactured by PerkinElmer
Sourced in United States
Tris-NaCl-blocking buffer is a laboratory reagent used to reduce non-specific binding in immunoassays and other protein-based applications. It contains Tris buffer and sodium chloride to maintain appropriate pH and ionic strength.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33258, by Merck Group (1 mentions)
E cadherin, by BD (1 mentions)
Evos fl auto imaging system, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit, by Abcam (1 mentions)
Histovt one, by Nacalai Tesque (1 mentions)
Envision system containing horseradish peroxidase, by Agilent Technologies (1 mentions)
Lsm 710 confocal laser scanning microscope, by Zeiss (1 mentions)
Bz x710 fluorescent microscope, by Keyence (1 mentions)
3 protocols using tris nacl blocking buffer
1
Immunocytochemical Analysis of iPSCs
2
Immunohistochemical Detection of Vaccinia Virus in Tumor Samples
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Tumors harvested at euthanasia were formalin fixed for 2 weeks, embedded in paraffin, and cut into 5-μm-thick sections. H&E staining was done for routine histopathological examination. Slides were deparaffinized and applied for heat-mediated antigen retrieval per manufacturer’s protocol (IHC World, Ellicott City, MD, USA). Tumor sections were then permeabilized with methanol, and Tris-NaCl-blocking buffer (PerkinElmer, Waltham, MA, USA) was used to decrease background for 20 min. Rabbit anti-vaccinia virus antibody 1:200 in Tris-NaCl-blocking buffer (category no. ab35219; Abcam, Cambridge, MA, USA) was added overnight in a humidified chamber at 4°C. The next day, tumor sections were secondarily stained with Alexa Fluor 488-conjugated goat anti-rabbit (category no. ab150080; Abcam, Cambridge, MA, USA) for 1 h at room temperature. Finally, DAPI was added for nuclear staining, and images were obtained using EVOS FL Auto Imaging System (Thermo Fisher Scientific, Waltham, MA, USA).
3
Immunohistochemistry for Protein Localization
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We performed immunohistochemistry as previously described (55 , 65 (link)). Briefly, the frozen sections were incubated for 20 min at 70°C in HistoVT One (Nacalai) for antigen retrieval. The paraffin sections were deparaffinized, rehydrated, and then heated in a microwave for 15 min in 10 mM citrate buffer at pH 6. The sections were incubated with Tris-NaCl-blocking buffer (PerkinElmer) for 20 min at room temperature, primary antibodies overnight at 4°C, and fluorescent secondary antibodies for 30 min at room temperature. Between all steps, the sections were washed with phosphate-buffered saline without detergent three times. See table S6 for a list of antibodies. An EnVision+ System containing horseradish peroxidase (Dako) was used for 3,3′-diaminobenzidine staining. Image data were collected using LSM710 laser scanning confocal microscope (Carl Zeiss) or BZ-X710 fluorescent microscope (Keyence).
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