MA5-12287

antibody from Invitrogen Antibodies

Targeting: CD99 MIC2

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Immunohistochemistry (Supportive data in Antibodypedia)

Flow cytometry (Supportive data in Antibodypedia)

Antibody Data

Product number

MA5-12287

Provider

Invitrogen Antibodies

Product name

CD99 Monoclonal Antibody (O13)

Antibody type

Monoclonal

Antigen

Other

Description

MA5-12287 targets CD99 in IHC (P) applications and shows reactivity with Human samples.

Antibody clone number

O13

Concentration

0.2 mg/mL

References

Submitted references

Clofarabine induces ERK/MSK/CREB activation through inhibiting CD99 on Ewing sarcoma cells.

Sevim H, Çelik H, Düşünceli L, Ceyhan CS, Molotkova A, Nakazawa K, Graham GT, Petro JR, Toretsky JA, Üren A
PloS one 2021;16(6):e0253170

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Histopathological, immunophenotypic and clinical particularities and evolution of a case of hepatosplenic T-cell lymphoma in transformation to leukemia.

Benedek Lázár E, Köpeczi JB, Tunyogi AB, Kakucs E, Horváth E, Turcu M, Benedek I
Romanian journal of morphology and embryology = Revue roumaine de morphologie et embryologie 2013;54(4):1135-9

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MiR-30a-5p connects EWS-FLI1 and CD99, two major therapeutic targets in Ewing tumor.

Franzetti GA, Laud-Duval K, Bellanger D, Stern MH, Sastre-Garau X, Delattre O
Oncogene 2013 Aug 15;32(33):3915-21

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Inducible expression of chimeric EWS/ETS proteins confers Ewing's family tumor-like phenotypes to human mesenchymal progenitor cells.

Miyagawa Y, Okita H, Nakaijima H, Horiuchi Y, Sato B, Taguchi T, Toyoda M, Katagiri YU, Fujimoto J, Hata J, Umezawa A, Kiyokawa N
Molecular and cellular biology 2008 Apr;28(7):2125-37

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Kit expression in spindle cell rhabdomyosarcoma can possibly create a different approach for its tumorigenesis and therapy.

Diniz G, Aktas S, Ortac R, Tunakan M, Unlu I, Vergin C
Pathology, research and practice 2006;202(9):671-7

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of CD99 was performed using membrane enriched extracts (30 µg lysate) of U 87-MG (Lane 1), THP-1 (Lane 2), K562 (Lane 3), HeLa (Lane 4), A549 (Lane 5), HEL 92.1.7 (Lane 6), Ramos (Lane 7), and Raji (Lane 8). The blots were probed with Anti-CD99 Mouse Monoclonal Antibody (Product # MA5-12287, 2 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.4 µg/mL, 1:2500 dilution). A 30 kDa band corresponding to CD99 was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10 % Bis-Tris gel (Product # NP0302BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of CD99 was performed using 90% confluent log phase MOLT-4 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with CD99/MIC2 (O13) Mouse Monoclonal Antibody (Product # MA5-12287) at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing localization in the membrane. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

Immunohistochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Formalin-fixed, paraffin-embedded human Ewing’s sarcoma stained with CD99 antibody using peroxidase-conjugate and AEC chromogen. Note membrane staining of tumor cells.

Flow cytometry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Flow cytometry analysis of CD99 was performed using Jurkat cells. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with CD99 Mouse Monoclonal Antibody (MA5-12287, red histogram) or with mouse isotype control (pink histogram) at 3-5 ug/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Rabbit Anti-Mouse Secondary Antibody (A11059) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control..