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Rat anti cd8a

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The Rat anti-CD8a is a laboratory reagent used for the detection and analysis of CD8a expression on cells. CD8a is a cell surface glycoprotein that is expressed on a subset of T lymphocytes, known as cytotoxic T cells. This antibody can be used in various immunological techniques, such as flow cytometry, to identify and characterize CD8a-positive cells.

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Lab products found in correlation

Rat anti cd4, by Thermo Fisher Scientific (3 mentions) Antibody mix 1, by Thermo Fisher Scientific (2 mentions) Antibody mix 2, by Novus Biologicals (2 mentions) Rat anti b220, by BioLegend (2 mentions) Anti rabbit 647, by Cell Signaling Technology (2 mentions) Anti rat 647, by Cell Signaling Technology (2 mentions) Ab15323, by Proteintech (2 mentions) 3 3 diaminobenzidine substrate, by Vector Laboratories (1 mentions) Rat anti foxp3, by Thermo Fisher Scientific (1 mentions) Immpress hrp goat anti rat igg polymer detection kit, by Vector Laboratories (1 mentions) Mayer s hematoxylin, by Thermo Fisher Scientific (1 mentions) Bx51 microscope, by Olympus (1 mentions) Rat anti cd3, by Thermo Fisher Scientific (1 mentions) Optilyse c solution, by Thermo Fisher Scientific (1 mentions) Anti rat cd25, by Thermo Fisher Scientific (1 mentions) Rat anti mouse foxp3, by Thermo Fisher Scientific (1 mentions) Flow cytometry, by Beckman Coulter (1 mentions) Las af software, by Leica camera (1 mentions) Hoechst 33258, by Merck Group (1 mentions) Confocal microscope, by Leica camera (1 mentions)

5 protocols using rat anti cd8a

1

Multiparametric Immunostaining of Murine Lung Sections

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Sequential immunostaining was performed on 3μm thick Formalin-Fixed Paraffin-Embedded (FFPE) murine lung sections. Briefly, heat mediated antigen retrieval was performed in citrate pH 6.0 (antibody Mix 1, ThermoFisher), or in EDTA pH 9.0 (antibody Mix 2, Novus) buffer. Antibody elution was performed between each staining cycle59 . Antibodies used in Mix 1 were; Rat anti-CD4 (1:200, ThermoFisher #14–9766-82), Rat anti-CD8a (1:200, ThermoFisher #14–0808-82) and Rat anti-B220 (1:500, Biolegend #103202). Antibodies used in Mix 2 were; Rabbit anti-IBA1 (1:1000, VWR #100369–764), Rabbit anti-iNOS (1:100, Abcam #ab15323) and Rabbit anti-CD206 (1:500, ProteinTech #18704–1-AP). Secondary antibodies used were: anti-Rabbit 555 (1:500, Cell Signaling #4413S), anti-Rabbit 647 (1:500, Cell Signaling #4414S) and anti-Rat 647 (1:500, Cell Signaling #4418S).
Acquired images were processed using FIJI and the FIJI plugin HyperStackReg V5.660 (and Ved Sharma. 2018, December 13). ImageJ plugin HyperStackReg V5.6 (Zenodo. http://doi.org/10.5281/zenodo.2252521). Autofluorescence acquired in non-relevant channels was substracted as appropriate. IBA1 and iNOS staining quantitation was performed using Ilastik (v1.3.3post2)61 and CellProfiler (v3.1.9)62 .
Conlon T.M., John-Schuster G., Heide D., Pfister D., Lehmann M., Hu Y., Ertüz Z., Lopez M.A., Ansari M., Strunz M., Mayr C., Ciminieri C., Costa R., Kohlhepp M.S., Guillot A., Günes G., Jeridi A., Funk M.C., Beroshvili G., Prokosch S., Hetzer J., Verleden S.E., Alsafadi H., Lindner M., Burgstaller G., Becker L., Irmler M., Dudek M., Janzen J., Goffin E., Gosens R., Knolle P., Pirotte B., Stoeger T., Beckers J., Wagner D., Singh I., Theis F.J., de Angelis M.H., O’Connor T.O., Tacke F., Boutros M., Dejardin E., Eickelberg O., Schiller H.B., Königshoff M., Heikenwalder M, & Yildirim A.Ö. (2020). Inhibition of LTβR-signalling activates Wnt-induced regeneration in lung. Nature, 588(7836), 151-156.
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2

Multiparametric Immunostaining of Murine Lung Sections

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Sequential immunostaining was performed on 3μm thick Formalin-Fixed Paraffin-Embedded (FFPE) murine lung sections. Briefly, heat mediated antigen retrieval was performed in citrate pH 6.0 (antibody Mix 1, ThermoFisher), or in EDTA pH 9.0 (antibody Mix 2, Novus) buffer. Antibody elution was performed between each staining cycle59 . Antibodies used in Mix 1 were; Rat anti-CD4 (1:200, ThermoFisher #14–9766-82), Rat anti-CD8a (1:200, ThermoFisher #14–0808-82) and Rat anti-B220 (1:500, Biolegend #103202). Antibodies used in Mix 2 were; Rabbit anti-IBA1 (1:1000, VWR #100369–764), Rabbit anti-iNOS (1:100, Abcam #ab15323) and Rabbit anti-CD206 (1:500, ProteinTech #18704–1-AP). Secondary antibodies used were: anti-Rabbit 555 (1:500, Cell Signaling #4413S), anti-Rabbit 647 (1:500, Cell Signaling #4414S) and anti-Rat 647 (1:500, Cell Signaling #4418S).
Acquired images were processed using FIJI and the FIJI plugin HyperStackReg V5.660 (and Ved Sharma. 2018, December 13). ImageJ plugin HyperStackReg V5.6 (Zenodo. http://doi.org/10.5281/zenodo.2252521). Autofluorescence acquired in non-relevant channels was substracted as appropriate. IBA1 and iNOS staining quantitation was performed using Ilastik (v1.3.3post2)61 and CellProfiler (v3.1.9)62 .
Conlon T.M., John-Schuster G., Heide D., Pfister D., Lehmann M., Hu Y., Ertüz Z., Lopez M.A., Ansari M., Strunz M., Mayr C., Ciminieri C., Costa R., Kohlhepp M.S., Guillot A., Günes G., Jeridi A., Funk M.C., Beroshvili G., Prokosch S., Hetzer J., Verleden S.E., Alsafadi H., Lindner M., Burgstaller G., Becker L., Irmler M., Dudek M., Janzen J., Goffin E., Gosens R., Knolle P., Pirotte B., Stoeger T., Beckers J., Wagner D., Singh I., Theis F.J., de Angelis M.H., O’Connor T.O., Tacke F., Boutros M., Dejardin E., Eickelberg O., Schiller H.B., Königshoff M., Heikenwalder M, & Yildirim A.Ö. (2020). Inhibition of LTβR-signalling activates Wnt-induced regeneration in lung. Nature, 588(7836), 151-156.
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3

Quantification of Immune Cell Populations in Tumor Tissue

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Animal procedures and maintenance were conducted in accordance with the University of Wisconsin-Madison School of Medicine and Public Health IACUC guidelines. Tumor tissue samples were collected, fixed in 10% neutral buffered formalin, and paraffin embedded. Antigen retrieval was performed by heating the sections in 10mM citrate buffer (pH 6.0) in a decloaking chamber. Samples were incubated with rat anti-CD8a (Thermo Fisher Scientific #14080882, 1:500), rat anti-CD4 (Thermo Fisher Scientific #14976682, 1:500), or rat anti-FoxP3 (Thermo Fisher Scientific #14577382, 1:200). Sections were stained using the ImmPRESS HRP Goat Anti-Rat IgG Polymer Detection Kit (Vector Laboratories, Burlingame, CA, #MP-7404–50). Antibody binding was revealed by the addition of 3,3’-diaminobenzidine substrate (Vector Laboratories). Tissues were counterstained with Mayer’s hematoxylin (Thermo Fisher Scientific). Tissues were examined using an Olympus BX51 microscope. Quantitation of staining intensity was performed as previously described26 using FIJI.
Kostecki K.L., Iida M., Wiley A.L., Kimani S., Mehall B., Tetreault K., Alexandridis R., Yu M., Hong S., Salgia R., Bruce J.Y., Birge R.B., Harari P, & Wheeler D.L. (2023). Dual Axl/MerTK inhibitor INCB081776 creates a pro-inflammatory tumor immune microenvironment and enhances anti-PDL1 efficacy in head and neck cancer. Head & neck, 45(5), 1255-1271.
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4

Lymphocyte Subpopulation Analysis

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Absolute and relative numbers of lymphocytes of various subpopulations in peripheral blood were counted using flow cytometry (Beckman Coulter). The following antibodies (eBioscience) were used for immune phenotypic analysis of the main subpopulations of lymphocytes: anti-rat CD3 (for T lymphocytes); anti-rat CD4 (for T helpers); anti-rat CD8a (for cytotoxic T cells); anti-rat CD45R (for B lymphocytes); anti-rat CD25 (for activated T cells); anti-mouse/rat Foxp3 (for regulatory T cells); and anti-rat CD314 (for NK cells). Erythrocytes were lysed with OptiLyse C solution (eBioscience).
Kosyreva A.M., Makarova O.V., Kakturskiy L.V., Mikhailova L.P., Boltovskaya M.N, & Rogov K.A. (2018). Sex differences of inflammation in target organs, induced by intraperitoneal injection of lipopolysaccharide, depend on its dose. Journal of Inflammation Research, 11, 431-445.
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5

Quantifying Tumor-Infiltrating CD8+ T Cells

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Dissected B16F10 mouse tumors were embedded in Tissue-Tek OCT and frozen in liquid nitrogen. Sectioned specimens were washed with 1× PBS (pH 6.8) and incubated with glycine (0.1 M) for 30 min followed by incubation with 1% BSA for 1 h. These samples were then incubated overnight with anti-rat CD8a (eBIOSCIENCES) with a 1:100 dilution at 4°C. The slides were subsequently washed and incubated with secondary anti-rat conjugated to FITC antibodies (Invitrogen) with a 1:500 dilution at room temperature for 1 h. Nuclear staining was performed using Hoechst 33258 (Sigma-Aldrich) according to the manufacturer’s instructions. Images were captured using a Leica Confocal Microscope and images were analyzed using LAS AF software (LEICA).
Manrique-Rincón A.J., Beraldo C.M., Toscaro J.M, & Bajgelman M.C. (2017). Exploring Synergy in Combinations of Tumor-Derived Vaccines That Harbor 4-1BBL, OX40L, and GM-CSF. Frontiers in Immunology, 8, 1150.
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