Biotinylated uea 1, by Vector Laboratories - Product details

1

Immunofluorescence Staining of Thymic Cryosections

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cryosections (5/6 μm thick) were stained as described previously.^{50 (link)} The primary antibodies used were rabbit anti-FoxN1 (provided by Dr. Itoi),^{43 (link)} anti-claudin-3,4 (Invitrogen, Grand Island, NY, USA, #34-1700 and #36-4800), anti-Keratin-5 (Covance, Princeton, NJ, USA, #PRB-160P), Biotinylated-UEA-1 (Vector Laboratories, Burlingame, CA, USA, #B-1065), anti-Aire (Santa Cruz, Dallas, TX, USA, #SC-33189, anti-β5t (Medical and Biological Laboratories, Nagoya, Japan, #PD021), and anti-Keratin-8 (Troma-1 supernatant). The secondary antibodies used were Cy3-conjugated or Alexa-Fluor-488-conjugated donkey anti-rabbit or -rat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA).

Ruan L., Zhang Z., Mu L., Burnley P., Wang L., Coder B., Zhuge Q, & Su D.M. (2014). Biological significance of FoxN1 gain-of-function mutations during T and B lymphopoiesis in juvenile mice. Cell Death & Disease, 5(10), e1457-.

+ Open protocol

+ Expand

2

Murine Immune Cell Isolation and Characterization

Check if the same lab product or an alternative is used in the 5 most similar protocols

C57BL/6 mice were purchased from Chubu Kagaku Shizai (Nagoya, Japan). All the mice were handled in accordance with the Guidelines for Animal Experiments, Gifu University (Gifu, Japan). Mice are sacrificed by cervical dislocation. The APC-Cy7-conjugated CD4, PE-Cy-7-conjugated CD8 antibodies, PE-conjugated CD80 antibody, the purified anti-mouse CD16/32, the APC-Cy7-conjugated rat anti-mouse CD45, APC-Cy7-conjugated TER-119, and PE-conjugated anti-mouse EpCAM (G8.8) were obtained from Biolegend (San Diego, CA). The rabbit anti-mouse keratin-5 antibody was purchased from Covance (Berkeley, CA). The biotinylated anti-mouse keratin-8 antibody was from PROGEN (Heidelberg, Germany). The biotinylated UEA-1 was obtained from VECTOR Laboratories (Burlingame, CA). Further, 7-aminoactinomycin D was purchased from Wako (Tokyo, Japan).

Tateishi R., Akiyama N., Miyauchi M., Yoshinaga R., Sasanuma H., Kudo T., Shimbo M., Shinohara M., Obata K., Inoue J.I., Shirakawa M., Shiba D., Asahara H., Yoshida N., Takahashi S., Morita H, & Akiyama T. (2015). Hypergravity Provokes a Temporary Reduction in CD4+CD8+ Thymocyte Number and a Persistent Decrease in Medullary Thymic Epithelial Cell Frequency in Mice. PLoS ONE, 10(10), e0141650.

+ Open protocol

+ Expand

3

Multiparametric Flow Cytometry Analysis of Immune Cell Populations

Check if the same lab product or an alternative is used in the 5 most similar protocols

Rabbit anti–mouse keratin-5 antibody was purchased from Covance. Biotinylated UEA-1 was obtained from Vector Laboratories. The APC-Cy7–conjugated rat anti–mouse CD45, PE-Cy7–conjugated TER119, and PE-conjugated anti–mouse EpCAM (G8.8) antibodies were obtained from BD or BioLegend. The PE-conjugated rat anti-CD45, PE-conjugated mouse TER-119, FITC-conjugated mouse MHC II (M5/114.15.1), and APC-conjugated Aire antibodies were obtained from eBioscience. PE-conjugated CD80, CD86, CD40, and PD-L1 antibodies, purified anti–mouse CD16/32, FITC-conjugated ant–mouse EpCAM (G8.8), APC-Cy7–conjugated CD4, PE–Cy-7–conjugated CD8, and APC-conjugated GITR antibodies were obtained from BioLegend. PE-conjugated CD8, FITC-conjugated CD4, and FITC-conjugated CD3e antibodies were obtained from BD. PE-conjugated anti-Foxp3 (FJK-16s) was purchased from eBioscience. 7-aminoactinomycin D and recombinant mouse RANKL were purchased from Wako Pure Chemical Industries. Anti–mouse OPG antibody was purchased from R&D Systems. A RANK-Fc chimera was obtained from Sigma-Aldrich. Alexa Fluor 546–conjugated streptavidin and Alexa Fluor 488–conjugated anti–rabbit IgG were obtained from Invitrogen.

Akiyama N., Shinzawa M., Miyauchi M., Yanai H., Tateishi R., Shimo Y., Ohshima D., Matsuo K., Sasaki I., Hoshino K., Wu G., Yagi S., Inoue J.I., Kaisho T, & Akiyama T. (2014). Limitation of immune tolerance–inducing thymic epithelial cell development by Spi-B–mediated negative feedback regulation. The Journal of Experimental Medicine, 211(12), 2425-2438.

+ Open protocol

+ Expand

4

Thymic Epithelial Cell Isolation and Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

For thymic epithelial cell analysis, single cell suspensions were generated by digesting thymic lobes with collagenase dispase (2.5mg/ml, Roche) and DNase 1 (40mg/ml Roche). CD45^- cells were enriched by the depletion of CD45⁺ cells using anti-CD45 beads and LS columns (Miltenyi Biotec). The following antibodies were used for TEC analysis: anti-CD45 clone 30-F11 (eBioscience), anti-EpCAM1 clone G8.8 (eBioscience), anti-Ly51 clone 6C3 (Biolegend), anti-MHCII clone M5/114.15.2 (eBioscience), anti-CD80 clone 16-10A1 (Biolegend), CD104 clone 346-11A (Biolegend), and anti-MHCI 28-14-8. Biotinylated UEA-1 (Vector laboratories) was detected using streptavidin PECy7 (eBioscience). Cells were analysed using a LSR Fortessa (Becton Dickinson) with data analysis carried out using Flowjo v10 (Becton Dickinson). For cell sorting, TEC subsets were identified using the antibodies above, and isolated using a FACS Aria Fusion 1 cell sorter (Becton Dickinson).
The sorting strategy for the different TEC subsets were as follows, Cxcl12^DsRed+ cTEC: CD45^-EpCAM1⁺UEA1^-Ly51⁺CXCL12^DsRed+; CXCL12^DsRed- cTEC: CD45^-EpCAM1⁺UEA1^-Ly51⁺CXCL12^DsRed-; mTEC^lo: CD45^-EpCAM1⁺UEA1⁺Ly51^-CD80^-MHCII^-; mTEC^hi: CD45^-EpCAM1⁺UEA-1^-Ly51⁺CD80⁺MHCII⁺; CD104⁺ mTEC^lo, CD45^-EpCAM1⁺UEA1⁺Ly51^-CD80^-MHCII^-CD104⁺; CD104^- mTEC^lo, CD45^-EpCAM1⁺UEA1⁺Ly51^-CD80^-MHCII^-CD104^-.

White A.J., Parnell S.M., Handel A., Maio S., Bacon A., Cosway E.J., Lucas B., James K.D., Cowan J.E., Jenkinson W.E., Hollander G.A, & Anderson G. (2023). Diversity in Cortical Thymic Epithelial Cells Occurs through Loss of a Foxn1-Dependent Gene Signature Driven by Stage-Specific Thymocyte Cross-Talk. The Journal of Immunology Author Choice, 210(1), 40-49.

+ Open protocol

+ Expand

5

Murine Immune Cell Profiling

Check if the same lab product or an alternative is used in the 5 most similar protocols

The following fluorescent anti–mouse antibodies were used for flow cytometry: anti-CD16/32, APCCy7-anti-CD45 (clone 30 F-11), APCCy7- or PECy7-anti-TER119 (clone TER-119), PE- or PECy7-anti-EpCAM (CD326, clone G8.8), FITC- or PECy7-anti-MHCII (I-A/I-E, clone M5/114.15.2), APC-anti-Ly51 (clone 6C3; BioLegend), PE-anti-CD80 (clone 16-10A1), PE-anti-CD24 (clone M1/69; eBioscience), and biotinylated UEA-1 (Vector Laboratories) antibodies. An agonistic anti-LtbR antibody was purchased from Alexis Biochemicals. Recombinant RANKL was a generous gift from Oriental Yeast Co. Anti-RANKL antibodies were prepared from a subclone of hybridoma obtained by fusing mouse myeloma cells with B cells.

Akiyama N., Takizawa N., Miyauchi M., Yanai H., Tateishi R., Shinzawa M., Yoshinaga R., Kurihara M., Demizu Y., Yasuda H., Yagi S., Wu G., Matsumoto M., Sakamoto R., Yoshida N., Penninger J.M., Kobayashi Y., Inoue J.I, & Akiyama T. (2016). Identification of embryonic precursor cells that differentiate into thymic epithelial cells expressing autoimmune regulator. The Journal of Experimental Medicine, 213(8), 1441-1458.

+ Open protocol

+ Expand

6

Multicolor Flow Cytometry and Immunohistochemistry

Check if the same lab product or an alternative is used in the 5 most similar protocols

The following antibodies were used for flow cytometric studies: anti-CD8 (53–6.7), anti-CD4 (H129.19), anti-CD3ε (145-2C11, 500A2), anti-CD62L (MEL-14), anti-CD44 (IM7), and anti-CD25 (PC61), anti-CD19 (1D3), Mac-1 (M1/70), γδ TCR (UC7-13D5) (all purchased from BD PharMingen, San Jose, CA). For immunohistochemistry, the following antibodies were used: Rabbit anti-cytokeratin 14 (K14; rabbit, COVANCE, Princeton, NJ), rabbit anti-K5 (rabbit, COVANCE, Princeton, NJ), and biotinylated mouse anti-K8 (PROGEN, Heidelberg, Germany), biotinylated UEA-1 (VECTOR LABORATORIES, Burlingame, CA), rabbit anti-β5t (MBL, Nagoya, Japan), ERTR5 [73 (link)]. Polyclonal anti-Aire antibody was a kind gift from M. Matsumoto (Tokushima Univ.). All secondary reagents for immunohistochemistry were purchased from Molecular Probes (Carlsbad, CA): Alexa Fluor488 donkey anti-rabbit IgG (H+L) conjugate, Alexa Fluor488 goat anti-rabbit IgG (H+L) conjugate, Alexa Fluor488 streptavidin conjugate, Alexa Fluor546 goat anti-rabbit IgG (H+L) conjugate and Alexa Fluor546 streptavidin conjugate.

Satoh R., Kakugawa K., Yasuda T., Yoshida H., Sibilia M., Katsura Y., Levi B., Abramson J., Koseki Y., Koseki H., van Ewijk W., Hollander G.A, & Kawamoto H. (2016). Requirement of Stat3 Signaling in the Postnatal Development of Thymic Medullary Epithelial Cells. PLoS Genetics, 12(1), e1005776.

+ Open protocol

+ Expand

7

Isolation and Flow Cytometry Analysis of Thymic Epithelial Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

For TEC analysis, single-cell suspensions were generated by digesting thymic lobes with collagenase Dispase (2.5 mg/ml; Roche) and DNase 1 (40 mg/ml; Roche). CD45⁻ cells were enriched by the depletion of CD45⁺ cells using anti-CD45 beads and LS columns (Miltenyi Biotec). The following Abs were used for TEC analysis: anti-CD45 clone 30-F11 (eBioscience), anti-EpCAM1 clone G8.8 (eBioscience), anti-Ly51 clone 6C3 (BioLegend), anti–MHC II clone M5/114.15.2 (eBioscience), anti-CD80 clone 16-10A1 (BioLegend), CD104 clone 346-11A (BioLegend), and anti–MHC I 28-14-8. Biotinylated UEA-1 (Vector laboratories) was detected using streptavidin PECy7 (eBioscience). Cells were analyzed using a LSR Fortessa (Becton Dickinson) with data analysis carried out using FlowJo v10 (Becton Dickinson). For cell sorting, TEC subsets were identified using the earlier Abs and isolated using a FACSAria Fusion 1 cell sorter (Becton Dickinson). The sorting strategy for the different TEC subsets was as follows: Cxcl12^DsRed+ cTEC, CD45⁻EpCAM1⁺UEA1⁻Ly51⁺CXCL12^DsRed+; CXCL12^DsRed− cTEC, CD45⁻EpCAM1⁺UEA1⁻Ly51⁺CXCL12^DsRed−; mTEC^lo, CD45⁻EpCAM1⁺UEA1⁺Ly51⁻CD80⁻MHC II⁻; mTEC^hi, CD45⁻EpCAM1⁺UEA⁺Ly51⁻CD80⁺MHC II⁺; CD104⁺ mTEC^lo, CD45⁻EpCAM1⁺UEA1⁺Ly51⁻CD80⁻MHC II⁻CD104⁺; and CD104⁻ mTEC^lo, CD45⁻EpCAM1⁺UEA1⁺Ly51⁻CD80⁻MHC II⁻CD104⁻.

White A.J., Parnell S.M., Handel A., Maio S., Bacon A., Cosway E.J., Lucas B., James K.D., Cowan J.E., Jenkinson W.E., Hollander G.A, & Anderson G. (2023). Diversity in Cortical Thymic Epithelial Cells Occurs through Loss of a Foxn1-Dependent Gene Signature Driven by Stage-Specific Thymocyte Cross-Talk. The Journal of Immunology Author Choice, 210(1), 40-49.

+ Open protocol

+ Expand

8

Thymus Tissue Immunofluorescence Staining

Check if the same lab product or an alternative is used in the 5 most similar protocols

Adult mice were transcardially perfused with PBS followed by 2% paraformaldehyde (PFA) in PBS under deep anesthesia with diethyl ether. The thymus was removed and fixed in 2% PFA in PBS for 2 h, immersed in sucrose gradient (10%, 20% and 30% wt/vol) in PBS sequentially 24 h for each step at 4 °C, then tissues were embedded in optimal cutting temperature compound (OCT). Frozen samples were sectioned at 6 μm. Cryosections were stained with Biotinylated UEA1 at 10 μg ml⁻¹ (Vector Laboratories), chicken anti-GFP at 10 μg ml⁻¹ (Abcam) and rabbit anti-RFP at 10 μg ml⁻¹ (Rockland) followed by Alexa Fluor 647-conjugated streptavidin (Thermo Fisher), goat anti-chicken IgY Alexa 488 (Abcam) and goat anti-rabbit Alexa 546 (Thermo Fisher).

Tai X., Indart A., Rojano M., Guo J., Apenes N., Kadakia T., Craveiro M., Alag A., Etzensperger R., Badr M.E., Zhang F., Zhang Z., Mu J., Guinter T., Crossman A., Granger L., Sharrow S., Zhou X, & Singer A. (2023). How autoreactive thymocytes differentiate into regulatory versus effector CD4+ T cells after avoiding clonal deletion. Nature Immunology, 24(4), 637-651.

+ Open protocol

+ Expand

9

Isolation and Flow Cytometry Analysis of Thymic Epithelial Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

For TEC analysis, single-cell suspensions were generated by digesting thymic lobes with collagenase Dispase (2.5 mg/ml; Roche) and DNase 1 (40 mg/ml; Roche). CD45⁻ cells were enriched by the depletion of CD45⁺ cells using anti-CD45 beads and LS columns (Miltenyi Biotec). The following Abs were used for TEC analysis: anti-CD45 clone 30-F11 (eBioscience), anti-EpCAM1 clone G8.8 (eBioscience), anti-Ly51 clone 6C3 (BioLegend), anti–MHC II clone M5/114.15.2 (eBioscience), anti-CD80 clone 16-10A1 (BioLegend), CD104 clone 346-11A (BioLegend), and anti–MHC I 28-14-8. Biotinylated UEA-1 (Vector laboratories) was detected using streptavidin PECy7 (eBioscience). Cells were analyzed using a LSR Fortessa (Becton Dickinson) with data analysis carried out using FlowJo v10 (Becton Dickinson). For cell sorting, TEC subsets were identified using the earlier Abs and isolated using a FACSAria Fusion 1 cell sorter (Becton Dickinson). The sorting strategy for the different TEC subsets was as follows: Cxcl12^DsRed+ cTEC, CD45⁻EpCAM1⁺UEA1⁻Ly51⁺CXCL12^DsRed+; CXCL12^DsRed− cTEC, CD45⁻EpCAM1⁺UEA1⁻Ly51⁺CXCL12^DsRed−; mTEC^lo, CD45⁻EpCAM1⁺UEA1⁺Ly51⁻CD80⁻MHC II⁻; mTEC^hi, CD45⁻EpCAM1⁺UEA⁺Ly51⁻CD80⁺MHC II⁺; CD104⁺ mTEC^lo, CD45⁻EpCAM1⁺UEA1⁺Ly51⁻CD80⁻MHC II⁻CD104⁺; and CD104⁻ mTEC^lo, CD45⁻EpCAM1⁺UEA1⁺Ly51⁻CD80⁻MHC II⁻CD104⁻.

White A.J., Parnell S.M., Handel A., Maio S., Bacon A., Cosway E.J., Lucas B., James K.D., Cowan J.E., Jenkinson W.E., Hollander G.A, & Anderson G. (2023). Diversity in Cortical Thymic Epithelial Cells Occurs through Loss of a Foxn1-Dependent Gene Signature Driven by Stage-Specific Thymocyte Cross-Talk. The Journal of Immunology Author Choice, 210(1), 40-49.

+ Open protocol

+ Expand

Biotinylated uea 1

Lab products found in correlation

Close spelling variants of this product

9 protocols using biotinylated uea 1

Immunofluorescence Staining of Thymic Cryosections

Murine Immune Cell Isolation and Characterization

Multiparametric Flow Cytometry Analysis of Immune Cell Populations

Thymic Epithelial Cell Isolation and Analysis

Murine Immune Cell Profiling

Multicolor Flow Cytometry and Immunohistochemistry

Isolation and Flow Cytometry Analysis of Thymic Epithelial Cells

Thymus Tissue Immunofluorescence Staining

Isolation and Flow Cytometry Analysis of Thymic Epithelial Cells

About PubCompare

Ready to get started?