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Nat105

Manufactured by Abcam
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NAT105 is a primary antibody that recognizes the nuclear antigen. It can be used in immunohistochemistry applications.

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Lab products found in correlation

A0072, by Agilent Technologies (3 mentions) Histofine simple stain max po, by Nichirei Biosciences (2 mentions) Bond polymer refine detection kit, by Leica Biosystems (2 mentions) Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (2 mentions) Vectashield hardset antifade mounting medium with dapi, by Vector Laboratories (2 mentions) Vector trueview autofluorescence quenching kit, by Vector Laboratories (2 mentions) Ep459y, by Abcam (2 mentions) Bz x710 fluorescence microscope, by Keyence (2 mentions) Ir503, by Agilent Technologies (1 mentions) Ir604, by Agilent Technologies (1 mentions) Pg m1, by Agilent Technologies (1 mentions) Epr19759, by Abcam (1 mentions) Autostainer plus, by Agilent Technologies (1 mentions) Dako autostainer, by Agilent Technologies (1 mentions) Rodent block m, by Biocare Medical (1 mentions) Ki 67 antigen, by Agilent Technologies (1 mentions) Benchmark xt, by Roche (1 mentions) Bx51 microscope, by Olympus (1 mentions) E1j2j, by Cell Signaling Technology (1 mentions) Pd l1 22c3 pharmdx kit, by Agilent Technologies (1 mentions)

21 protocols using nat105

1

Immunoprofiling of Renal Cell Carcinoma

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A tissue microarray (TMA) containing 756 primary ccRCC and corresponding normal tissue samples was used as described before [28] (link). For immunohistochemical staining of tissue, microarray slides or formalin fixed and paraffin-embedded tumor tissue antibodies directed against CD3 (IR503, Dako, Glostrup, Denmark), CD8 (C8/144B, Dako), CD20 (IR604, Dako), CD56 (123C3, Dako), CD68 (PG-M1, Dako), CD138 (MI15, Dako), FoxP3 (236A/E7, Abcam, Cambridge, United Kingdom), myeloperoxidase (MPO) (IR511, Dako), PD-1 (NAT105, Abcam), and PD-L1 (EPR19759, Abcam) were used. All slides were stained with automatized immunostainers (autostainer plus, Dako).
Stenzel P.J., Schindeldecker M., Tagscherer K.E., Foersch S., Herpel E., Hohenfellner M., Hatiboglu G., Alt J., Thomas C., Haferkamp A., Roth W, & Macher-Goeppinger S. (2019). Prognostic and Predictive Value of Tumor-infiltrating Leukocytes and of Immune Checkpoint Molecules PD1 and PDL1 in Clear Cell Renal Cell Carcinoma. Translational Oncology, 13(2), 336-345.
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2

Comprehensive Tumor Histology Analysis

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Tumor histology was evaluated through H&E staining on a Dako autostainer (Agilent). Three micrometer sections from formalin-fixed, paraffin-embedded (FFPE) HT29 tumor were incubated with the appropriate serum designed for blocking endogenous mouse IgG and non-specific background in mouse tissues (Rodent Block M; Biocare Medical), and then incubated overnight at 4ºC using primary antibodies: anti-Ki-67 (1:75, Ki-67 Antigen (Dako Omnis) Clone MIB-1); cleaved caspase-3 antibody (1:250, Monoclonal Rabbit IgG Clone #269518 anti-human cleaved caspase-3 (Asp175) antibody); mouse monoclonal anti-human PD-1 (1:50 [NAT105] Abcam), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:400, D13.14.4E, XP Rabbit mAb #4370-CST), phospho-p38 MAPK (Thr180/Tyr182) (1:400, D3F9, XP Rabbit mAb #4511-CST). Anti-PD-1, predilute, NAT105 (Cell marque) in a Benchmark XT (Ventana Medical Systems) was used for section from patients with colon cancer. The IHC staining was evaluated in at least 10 consecutive not overlapping high-power field (HPF) ×400 magnification (0.237 mm2/field) in at least five areas using an Olympus BX51 microscope (Olympus, Tokyo, Japan). Stained sections were independently evaluated by expert pathologist/researchers blinded to initial assessments.
Ieranò C., Righelli D., D'Alterio C., Napolitano M., Portella L., Rea G., Auletta F., Santagata S., Trotta A.M., Guardascione G., Liotti F., Prevete N., Maiolino P., Luciano A., Barbieri A., Di Mauro A., Roma C., Esposito Abate R., Tatangelo F., Pacelli R., Normanno N., Melillo R.M, & Scala S. (2022). In PD-1+ human colon cancer cells NIVOLUMAB promotes survival and could protect tumor cells from conventional therapies. Journal for Immunotherapy of Cancer, 10(3), e004032.
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3

PD-L1, CD8, and HLA Expression in LUSC

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IHC staining was performed on representative surgical tissue sections from FFPE tissue blocks of LUSC patients using anti–human PD-L1 antibodies: E1J2J, Cell Signaling Technology; 28-8, Abcam; 22C3, Dako; SP142, Spring Bioscience; anti–human CD8 antibodies (C8/144B; Nichirei Bioscience); anti–human PD-1 antibodies (NAT105; Abcam); anti–human β2M antibodies (A0072; Dako); and anti–human HLA class I-A/B/C antibodies (EMR8-5; Hokudo). The detection of immunostaining was performed using Histofine Simple Stain MAX-PO (Nichirei Bioscience) or BOND polymer Refine Detection kit (Leica Biosystems). Some of the sections were stained with the PD-L1 22C3 pharmDx kit (Dako). PD-L1 positivity was defined as membranous or cytoplasmic staining in at least 1% of tumor cells. The staining results of β2M and HLA class I-A/B/C of tumor cells were categorized as negative (0%), focally positive (<50%), or positive (≥50%).
Sagawa R., Sakata S., Gong B., Seto Y., Takemoto A., Takagi S., Ninomiya H., Yanagitani N., Nakao M., Mun M., Uchibori K., Nishio M., Miyazaki Y., Shiraishi Y., Ogawa S., Kataoka K., Fujita N., Takeuchi K, & Katayama R. (2022). Soluble PD-L1 works as a decoy in lung cancer immunotherapy via alternative polyadenylation. JCI Insight, 7(1), e153323.
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4

Multiplex IHC for Immune Profiling

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IHC staining was performed on representative tissue sections from formalin-fixed and paraffin-embedded tissue blocks using anti-human CD8 antibodies (C8/144B; Nichirei Biosciences), anti-human PD-1 antibodies (NAT105; Abcam), anti-human HLA class I-A, B, and C antibodies (EMR8-5; HoKudo), anti-human B2M antibodies (A0072; Dako), anti-mouse CD8 antibodies (EPR20305; Abcam), anti-mouse PD-1 antibodies (D7D5W; Cell Signaling Technology), and anti-mouse granzyme B antibodies (D6E9W; Cell Signaling Technology).
Gong B., Kiyotani K., Sakata S., Nagano S., Kumehara S., Baba S., Besse B., Yanagitani N., Friboulet L., Nishio M., Takeuchi K., Kawamoto H., Fujita N, & Katayama R. (2019). Secreted PD-L1 variants mediate resistance to PD-L1 blockade therapy in non–small cell lung cancer. The Journal of Experimental Medicine, 216(4), 982-1000.
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5

Comprehensive Immunohistochemistry Profiling

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Tissue sections were deparaffinized in xylene and rehydrated through a graded ethanol series. Heat‐induced antigen retrieval was carried out in a high or low‐pH antigen retrieval buffer (DakoCytomation, Glostrup, Denmark). Endogenous peroxidase was blocked by incubating tissue sections in 3% H2O2 for 5 minutes. The primary antibodies against CD4 as a marker for helper T cells (1:500, EPR6855; Abcam, Cambridge, UK), CD8 as a maker for cytotoxic T lymphocytes (1:500, EP1150Y; Abcam), CD20 as a marker for pan B cells (1:50, L26; Abcam), Foxp3 as a marker for regulatory T cells (Treg) (1:300, 236A/E7; Abcam), PNAd as a marker for HEV (1:100, MECA‐79; BD PharmingenTM), programmed cell death (PD)‐1 as a marker for immune checkpoint molecules (1:50, NAT105; Abcam), Ki‐67 as a marker for cell proliferation (1:100, SP6; Abcam), CD80 as a marker for M1 macrophages (1:1000, EPR1157(2); Abcam) and CD163 as a marker for M2 macrophages (1:500, EPR11598; Abcam) were applied for 30 minutes. These sections were visualized using the HRP‐labeled polymer method (EnVision FLEX System, Dako). Immunostained sections were counterstained with hematoxylin, dehydrated in ethanol, and cleared in xylene. These data are summarized in Table S1.
Kuwabara S., Tsuchikawa T., Nakamura T., Hatanaka Y., Hatanaka K.C., Sasaki K., Ono M., Umemoto K., Suzuki T., Sato O., Hane Y., Nakanishi Y., Asano T., Ebihara Y., Kurashima Y., Noji T., Murakami S., Okamura K., Shichinohe T, & Hirano S. (2019). Prognostic relevance of tertiary lymphoid organs following neoadjuvant chemoradiotherapy in pancreatic ductal adenocarcinoma. Cancer Science, 110(6), 1853-1862.
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6

Immunohistochemical Profiling of Tumor Immune Markers

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From all included patients, formalin-fixed, paraffin-embedded (FFPE) pre-treatment biopsies were collected. Sections of the FFPE blocks were stained with hematoxylin and eosin (H&E) and assessed by a dedicated head and neck pathologist (S.M. Willems) to mark representative tumor regions. For each patient, three 0.6 mm tissue cores were obtained from the assigned area of the FFPE blocks and collected in a tissue microarray (TMA). The TMA was constructed by a fully automated tissue microarray instrument, as described before [22 (link)].
TMA tissue sections (4 µm) were immunohistochemically stained with antibodies for the following antigens: CD3 (A452; 1:200; DAKO), CD4 (SP35, 1:25; Cellmarque), CD8 (CD8/144B; 1:100; DAKO), FoxP3 (236A/E7; 1:750; Abcam), PD1 (NAT105; 1:100; Abcam), and PD-L1 (SP263, Ventana RTU). Staining was performed using a Ventana Bench Mark XT Autostainer (Ventana Medical Systems, Tucson, AZ, USA).
de Ruiter E.J., de Roest R.H., Brakenhoff R.H., Leemans C.R., de Bree R., Terhaard C.H, & Willems S.M. (2020). Digital pathology-aided assessment of tumor-infiltrating T lymphocytes in advanced stage, HPV-negative head and neck tumors. Cancer Immunology, Immunotherapy, 69(4), 581-591.
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7

Immunohistochemical Profiling of Immune Markers

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Formalin-fixed, paraffin-embedded specimens were stained with hematoxylin and eosin, CD4 (clone EP204, Sigma-Aldrich, St. Louis, MO), and CD8 (clone 4B11, Leica, Wetzlar, Germany) using standard automated protocols. Immunohistochemistry (IHC) for PD-1 (clone NAT105, Abcam, Cambridge, U.K.)16 (link) and PD-L1 (clone SP142, Spring Bioscience, Pleasanton, CA)17 (link) were performed as previously described, including the use of an isotype control for PD-L1 to control for potential false-positive staining.18 (link) Eight patient samples from the control and iLTS cohorts were stained for CD4, CD8, PD-1, and PD-L1. Eight iSGS patient samples were stained for only CD4, PD-1, and PD-L1 due to limited sample supply.
Davis R.J., Lina I., Ding D., Engle E.L., Taube J., Gelbard A, & Hillel A.T. (2020). Increased Expression of PD-1 and PD-L1 in Patients With Laryngotracheal Stenosis. The Laryngoscope, 131(5), 967-974.
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8

Evaluating PD-1 and PD-L1 Expression in Tumor Samples

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Formalin-fixed, paraffin-embedded tissue specimens obtained from surgical resections were examined. PD-1 and PD-L1 expression patterns were assessed by IHC on tumor samples by a pathologist blinded to clinical outcomes. The PD-1 antibody used was NAT105 (1:100, Abcam, Cambridge, United Kingdom) and PD-L1 antibody used was 22C3 (Merck Research Laboratories, Palo Alto, California). Expression was graded in a semiquantitative scoring system from 0 to 5 (0 indicating no expression, 5 indicating high expression) as described elsewhere.28 (link)–30
Owen D., Chu B., Lehman A.M., Annamalai L., Yearley J.H., Shilo K, & Otterson G.A. (2018). Expression Patterns, Prognostic Value, and Intratumoral Heterogeneity of PD-L1 and PD-1 in Thymoma and Thymic Carcinoma. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer, 13(8), 1204-1212.
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9

Multiplex Immunofluorescence Tissue Imaging

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Formalin-fixed, paraffin embedded 5 μm sections were deparaffinized and decloaked in 1% citrate buffer at pH 6.0 (Vector Laboratories). Sections were blocked in 1%BSA with goat serum prior to staining for CD3 (I nvitrogen, SP7, 1:150), PD-1 (Abcam, NAT105, 1:200), and CD20 (Abcam, EP459Y, 1:300) overnight. Following primary staining, tissues were washed and incubated with goat anti- mouse Alexa Fluor 488 (Invitrogen, 1:500) and goat anti- rabbit Alexa Fluor 594 (Invitrogen, 1:500) for 1 hour at room temperature. Background signal was quenched by 1-minute incubation using Vector TRUEView Autofluorescence Quenching Kit (Vector Laboratories). Coverslips were mounted and nuclei stained using Vectashield Hardset Antifade Mounting Medium with DAPI (Vector Laboratories). Composite tissue images were acquired on a Keyence BZ-X710 fluorescence microscope at 20X magnification. Identification of active germinal centers and T cell quantification were performed using open-source packages available in FiJI.
Smedley J.V., Bochart R.M., Fischer M., Funderburgh H., Kelly V., Crank H., Armantrout K., Shiel O., Robertson‐LeVay M., Sternberger N., Schmaling B., Roberts S., Sekiguchi V., Reusz M., Schwartz T., Meyer K.A., Webb G., Gilbride R.M., Dambrauskas N., Andrade D., Wood M., Labriola C., Axthelm M., Derby N., Varco‐Merth B., Fukazawa Y., Hansen S., Sacha J.B., Sodora D.L, & Sather D.N. (2022). Optimization and use of near infrared imaging to guide lymph node collection in rhesus macaques (Macaca mulatta). Journal of Medical Primatology, 51(5), 270-277.
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10

Immunohistochemical Analysis of Tumor Microenvironment

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Immunohistochemical analyses were performed, as previously described (5 (link),7 (link),8 (link)). The following antibody were used: anti-VEGF-A (1:200, JH121, Merck Millipore), anti-VEGF receptor (VEGFR)1 (1:200, AF321, R&D SYSTEMS), anti-VEGFR2 (1:600, 55B11, Cell Signaling Technology), anti-CD34 (1:100, F1604, Nichirei Biosciences Inc.), anti-HIF-1α (1:100, H-206, Santa Cruz Biotechnology), anti-carbonic anhydrase 9 (CA9) (1:50, H-120, Santa Cruz Biotechnology), anti-nestin (1:100, 10C2, Chemicon), anti-PD-1 (1:50, NAT105, Abcam), anti-PD-L1 (1:500, 28-8, Abcam), anti-CD4 (1:200, 1F6, Nichirei Bioscience Inc.), anti-CD8 (1:100, 144B, Abcam), anti-Foxp3 (1:100, ab54501, Abcam), and anti-CD163 (1:100, ab87099, Abcam). The status of the TME after Bev administration in our previous studies (5 (link)) was also highlighted using the original figures (Figure 1B). The results of the immunohistochemical analysis were evaluated, as previously described (5 (link),7 (link),8 (link)) (noted in Table 1).
Tamura R., Tanaka T., Morimoto Y., Kuranari Y., Yamamoto Y., Takei J., Murayama Y., Yoshida K, & Sasaki H. (2020). Alterations of the tumor microenvironment in glioblastoma following radiation and temozolomide with or without bevacizumab. Annals of Translational Medicine, 8(6), 297.
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