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Ab16669

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The Ab16669 is a lab equipment product from Thermo Fisher Scientific. It is designed for general laboratory use. No further details about its core function or intended use can be provided in an unbiased and factual manner.

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Lab products found in correlation

Arginase 1, by Cell Signaling Technology (2 mentions) Nanozoomer 2.0 ht, by Meyer Instruments (2 mentions) Npd view2, by Hamamatsu Photonics (2 mentions) Lsm 780, by Zeiss (2 mentions) Ab214421, by Abcam (1 mentions) Ba 9401, by Vector Laboratories (1 mentions) Clone 4sm95, by Thermo Fisher Scientific (1 mentions) Clone fjk 16s, by Thermo Fisher Scientific (1 mentions) Ba 1100, by Vector Laboratories (1 mentions) Alexa flour 594, by Thermo Fisher Scientific (1 mentions) Anti cd8a, by Thermo Fisher Scientific (1 mentions) Anti foxp3, by Thermo Fisher Scientific (1 mentions) Anti cd4, by Thermo Fisher Scientific (1 mentions) Anti rabbit texas red, by Thermo Fisher Scientific (1 mentions) Ab70369, by Abcam (1 mentions) Microtome, by Leica (1 mentions) Ab2893, by Abcam (1 mentions) Ab133357, by Abcam (1 mentions) Ab182973, by Abcam (1 mentions) Ab10558, by Abcam (1 mentions)

7 protocols using ab16669

1

Immunohistochemical Analysis of Tumor Samples

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Hematoxylin staining and immunostaining were performed on paraffin sections of tumors. Antibodies used were against LILRB4 (lab produced, 1:100), CD3 (Abcam, ab16669, 1:100), PD-1 (Thermo Fisher, J116, 14–9989-82, 1:100) and Arginase-1 (Cell signaling, 9819S, 1:100). The images were visualized using the Hamamatsu NanoZoomer 2.0-HT (Meyer instruments Inc., Houston, TX) and viewed in NPDview2 software (Hamamatsu, Japan).
Deng M., Gui X., Kim J., Xie L., Chen W., Li Z., He L., Chen Y., Chen H., Luo W., Lu Z., Xie J., Churchill H., Xu Y., Zhou Z., Wu G., Yu C., John S., Hirayasu K., Nguyen N., Liu X., Huang F., Li L., Deng H., Tang H., Sadek A.H., Zhang L., Huang T., Zou Y., Chen B., Zhu H., Arase H., Xia N., Jiang Y., Collins R., You M.J., Homsi J., Unni N., Lewis C., Chen G.Q., Fu Y.X., Liao X.C., An Z., Zheng J., Zhang N, & Zhang C.C. (2018). LILRB4 signaling in leukemia cells mediates T cell suppression and tumor infiltration. Nature, 562(7728), 605-609.
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2

Quantifying Immune Cell Populations in Intestinal Tumors

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Immunohistochemistry staining was performed on formalin-fixed, paraffin-embedded tissues from intestinal tumors taken from VCMsh2 mice treated with naproxen with or without the tetravalent FSPs vaccine according to standard protocols (see Supplementary Methods). Following primary antibodies were used: for CD3: clone SP7 abcam ab16669; for CD4: clone 4SM95 ThermoFisher Scientific #14–9766-82; for CD8: clone 4SM15 ThermoFisher Scientific #14–0808-82; for Foxp3: clone FJK-16s ThermoFisher Scientific #14–5773-82; for PD-1: clone EPR20665 abcam ab214421. Following biotinylated secondary antibodies were used: for CD3 and PD-1: Vector Laboratories BA-1100; for CD4, CD8 and Foxp3: Vector Laboratories BA-9401.
Zazzi M., Romano L., Catucci M., Venturi G., De Milito A, & Valensin P.E. (1999). Clinical Evaluation of an In-House Reverse Transcription-Competitive PCR for Quantitation of Human Immunodeficiency Virus Type 1 RNA in Plasma. Journal of Clinical Microbiology, 37(2), 333-338.
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3

Immunofluorescence Staining of Immune Cells

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We adapted the immunofluorescence (IF) staining protocol in this study from Cheng et al.17 (link) Briefly, tissues were dehydrated, paraffin-embedded, and cut into 5-μM thick sections that were mounted onto slides. Sections were deparaffinized and blocked with 5% goat serum in PBS for 1 h at room temperature. Antibodies were diluted in PBS with 1% bovine serum albumin. Anti-CD3+ (Abcam ab16669), anti-CD4+(Thermo Fisher MA1-146, Carlsbad, CA), anti-CD8a+(eBioscience 45-0081-80), anti-CD25+ (Thermo Fisher MA512680) and anti-FoxP3+ (Thermo Fisher 14-5773-82), anti-rabbit Texas Red (Thermo Fisher T2767), anti-rat Texas Red (Thermo Fisher T-6392), and anti-mouse Alexa Flour 594 (Thermo Fisher A-11032) were used for IF staining.
Wang X., Balaji S., Steen E.H., Li H., Rae M.M., Blum A.J., Miao Q., Butte M.J., Bollyky P.L, & Keswani S.G. (2019). T Lymphocytes Attenuate Dermal Scarring by Regulating Inflammation, Neovascularization, and Extracellular Matrix Remodeling. Advances in Wound Care, 8(11), 527-537.
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4

Immunohistochemical Staining of Cellular Markers

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For immunohistochemical analysis, samples were embedded in paraffin and sectioned at 6 μm using a microtome (Leica). Sections were deparaffinized, rehydrated and subjected to high-temperature antigen retrieval at pH 6 for all stains except for pKAP1 at pH 9. Detection of primary antibodies was performed using peroxidase-coupled appropriate secondary antibodies. Sections were counterstained with hematoxylin. Staining was imaged using a Pannoramic DESK scanner and analysed using QuPath67 (link). Due to the shape of macrophages (Supplementary Fig. 6), proper cell detection was not possible and mean intensity was used for the analysis. For histology, the following antibodies were used: anti-CD3 (Abcam Cat# ab16669, RRID:AB_443425, 1:100), anti-CD4 (Thermo Fisher Scientific Cat# 14-9766-82, RRID:AB_2573008, 1:100), anti-CD8 (Lab Vision Cat# RB-9009-P1, RRID:AB_149750, 1:100), anti-F4/80 (Abcam Cat# ab6640, RRID:AB_1140040, 1:1,000), anti-CD45R/B220 (BD Biosciences Cat# 550286, RRID:AB_393581, 1:50), anti-KAP1 phospho-S824 (Abcam Cat# ab70369, RRID:AB_1209417, 1:500), anti-γ-H2AX (Abcam Cat# ab2893, RRID:AB_303388, 1:1,000).
Gaballa A., Gebhardt-Wolf A., Krenz B., Mattavelli G., John M., Cossa G., Andreani S., Schülein-Völk C., Montesinos F., Vidal R., Kastner C., Ade C.P., Kneitz B., Gasteiger G., Gallant P., Rosenfeldt M., Riedel A, & Eilers M. (2024). PAF1c links S-phase progression to immune evasion and MYC function in pancreatic carcinoma. Nature Communications, 15, 1446.
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5

Immunohistochemical Analysis of Tumor Samples

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Hematoxylin staining and immunostaining were performed on paraffin sections of tumors. Antibodies used were against LILRB4 (lab produced, 1:100), CD3 (Abcam, ab16669, 1:100), PD-1 (Thermo Fisher, J116, 14–9989-82, 1:100) and Arginase-1 (Cell signaling, 9819S, 1:100). The images were visualized using the Hamamatsu NanoZoomer 2.0-HT (Meyer instruments Inc., Houston, TX) and viewed in NPDview2 software (Hamamatsu, Japan).
Deng M., Gui X., Kim J., Xie L., Chen W., Li Z., He L., Chen Y., Chen H., Luo W., Lu Z., Xie J., Churchill H., Xu Y., Zhou Z., Wu G., Yu C., John S., Hirayasu K., Nguyen N., Liu X., Huang F., Li L., Deng H., Tang H., Sadek A.H., Zhang L., Huang T., Zou Y., Chen B., Zhu H., Arase H., Xia N., Jiang Y., Collins R., You M.J., Homsi J., Unni N., Lewis C., Chen G.Q., Fu Y.X., Liao X.C., An Z., Zheng J., Zhang N, & Zhang C.C. (2018). LILRB4 signaling in leukemia cells mediates T cell suppression and tumor infiltration. Nature, 562(7728), 605-609.
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6

Western Blot Analysis of Immune Markers

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Protein was extracted as recently described (Zwischenberger et al., 2020). Protein isolates (5–10 μg) were resolved by SDS-PAGE electrophoresis (Bio-Rad Mini PROTEAN Tetra system) using TGX stain-free gradient (4–15%) gels, total protein was visualized using stain-free technology (ChemiDoc MP imaging system), and proteins were electrophoretically transferred (Bio-Rad Trans-blot Turbo Transfer System) to polyvinylidenedifluoride (pvdf) membranes. The membranes were blocked for 1 hr in 3% non-fat dry milk at room temperature, and incubated in primary antibody in 1% non-fat dry milk overnight at 4°C. HRP-linked secondary antibody incubation (Santa Cruz #SC-2004, 1:10,000 dilution) was conducted for 1 hr at room temperature, and the reaction was detected by chemiluminescence (Bio-Rad Clarity Western ECL Substrate). Primary antibodies were as follows: anti-PAI-1 antibody (Abcam ab182973, 1:5,000), anti-CD45 (Abcam, ab10558, 1:1,000), anti-CD11b (Abcam ab133357, 1:1,000), anti-CD3 (Abcam ab16669, 1:500), anti-CD31 (Invitrogen PA5-16301, 1:500), anti-CD34 (Invitrogen PA5-89536, 1:500). Densitometry analysis was performed on the resulting blots using Image Lab software, and normalized by total protein analysis.
Bruno M.E., Mukherjee S., Stromberg A.J., Saito H, & Starr M.E. (2021). Visceral fat-specific regulation of plasminogen activator inhibitor-1 in aged septic mice. Journal of cellular physiology, 237(1), 706-719.
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7

Comprehensive Kidney Histology and Immunofluorescence

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For conventional staining kidneys were perfused with ice-cold PBS and embedded in paraffin after overnight fixation in 4% paraformaldehyde (PFA) at 4 °C. Sections were cut at 2 µm and stained with hematoxylin and eosin, or PAS staining at the histology core of the USC Department of Pathology, Los Angeles, CA. For immunofluorescence, PFA fixed tissues were equilibrated in 30% sucrose/PBS overnight then embedded in OCT in dry ice ethanol bath. 8–10 µm frozen sections were washed in PBT (PBS + 1% Triton-X), blocked in 5% Normal Donkey Serum in PBT, and incubated overnight at 4 °C with primary antibodies and detected with species-specific secondary antibodies coupled to Alexa Fluor 488, 555, 594, and 647 (Life Technologies) for 1 h at room temperature. Following antibodies were used in this study to recognize CD3 (rabbit, Abcam, ab16669), CD19 (rat, Invitrogen, 13-0194-82), CD21 (rabbit, Abcam, ab75985), CD31 (rat, BD, 553370), CD45 (goat, BD, 550280), CD45R (rat, BD, 557390), Cxcl13 (goat, R&D, AF470), F4/80 (rat, eBioscience, 14–4801), Havcr1 (goat, R&D, AF1817), Ltl lectin-FITC conjugate (Vector Laboratories, FL-1321), Lyve1 (goat, R&D, AF2125), Ki67 (rabbit, Novocastra, Ki67p-CE). All sections were stained with Hoechst 33342 (Life Technologies) prior to mounting with Immu-Mount (Fisher). All images were acquired on Zeiss Axio Scan Z1 slide scanner and Zeiss LSM780.
Cippà P.E., Liu J., Sun B., Kumar S., Naesens M, & McMahon A.P. (2019). A late B lymphocyte action in dysfunctional tissue repair following kidney injury and transplantation. Nature Communications, 10, 1157.
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