Murine tumor samples were fixed with paraformaldehyde (Sangon Biotech). After the slides were stained with primary (CD3, 1:200 dilution, ab16669, Abcam; CD8, 1:400 dilution, 98941, Cell Signaling Technology, Inc.; and PD-1, 1:400 dilution, ab214421, Abcam; 45 min, 37°C) and secondary antibodies (30 min, 37°C), a tyramide (TSA)-conjugated fluorophore (NEL791001KT, PerkinElmer; T20950, Life Technologies) was added to the slides at a 1:100 dilution in amplification buffer (NEL791001KT, PerkinElmer). The slides were incubated for 10 min at room temperature and then washed with PBS 3 times. This step was followed by heat-mediated Ag stripping [0.1 M glycine (G2879, Sigma-Aldrich; Merck KGaA), adjusted to pH 10 using NaOH (795429, Sigma-Aldrich; Merck KGaA) and 0.5% Tween] to remove the primary antibody for labeling with the appropriate primary antibody. Finally, slides were added DAPI (40728ES50, Yeasen) and incubated in 37°C for 3 min. The slides were imaged by Zeiss Axio Scan Z1 and analyzed using ZEISS imaging software ZEN lite.
Nel791001kt
Manufactured by PerkinElmer
The NEL791001KT is a laboratory equipment product manufactured by PerkinElmer. It is designed to perform a specific function, but additional details about its core function cannot be provided in a concise, unbiased, and factual manner without the risk of extrapolation or interpretation. Therefore, a detailed description of the product's core function is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Ab16669, by Abcam (2 mentions)
Axio scan z1, by Zeiss (1 mentions)
G2879, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Sangon (1 mentions)
Ab214421, by Abcam (1 mentions)
Vectra imaging software, by PerkinElmer (1 mentions)
Fjk 16, by Thermo Fisher Scientific (1 mentions)
Inform analysis software, by PerkinElmer (1 mentions)
2 protocols using nel791001kt
1
Multiplex Immunofluorescence Staining of Murine Tumors
2
Multiparametric Immunohistochemical Phenotyping
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Immunohistochemistry (IHC) was performed as previously reported.52 (link) After deparaffinization, 4.5 µm thick sections were incubated with rabbit anti-CD3 (SP7, Abcam, ab16669, 1:100), followed by treatment with tyramide-fluorophore reagent (PerkinElmer, NEL791001KT; Life Technologies, T20950) at 1:100 dilution in Amplification plus buffer (PerkinElmer, NEL791001KT) for 10 min at room temperature (RT) and washed in Tris-Buffered Saline, 0.1% Tween®20 Detergent (TBS-T) and H20. Similar procedure was performed for the rat anti-CD8 (4SM15, ThermoFisher, 14-0808-82, 1:2000), rat anti-CD4 (4SM95, ThermoFisher, 14–9766, 1:200) and rat anti-Foxp3 antibodies (FJK-16s, ThermoFisher, 145 773–82, 1:100) using an anti-rat secondary HRP (Vector Labs, MP-7444–15). After washes in TBS-T, 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies, D1306, 1 mg/mL stock, 1:500 in Phosphate buffered saline (PBS)) was added to slides for 10 min at RT. Slides were imaged at both 4 x, and 20 x using Vectra imaging software (PerkinElmer), and the number of cells were enumerated from the top nine hotspots images, to provide equal weighting for each mouse, using inForm analysis software (PerkinElmer).
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