Ab185333

Manufactured by Abcam

Ab185333 is a laboratory reagent. It is a purified monoclonal antibody. The core function of this product is to bind to a specific target molecule.

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Lab products found in correlation

4 protocols using ab185333

Immunocytochemistry Staining of Microglia

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Cells were washed three times with DPBS (1X) and fixed with cold PFA (4% w/v) for 20 min at room temperature (RT) followed by three washes with PBS (1X). Cells were blocked with blocking solution (PBS with 0.1% Triton X-100 and 1% BSA) for 30 min at RT. Primary antibodies were added at respective dilutions (see below) in blocking solution and placed at 4°C overnight. The next day, cells were washed 3 times with PBS for 5 min and stained with Alexa Fluor conjugated secondary antibodies from Invitrogen (1:800) for 2.5 h at room temperature in the dark. After secondary staining, cells were washed 3 times with PBS and cover slipped with ProLong™ Diamond Antifade Mountant or Fluoromount-G™ (ThermoFisher). Primary antibodies used for ICC: anti-P2ry12 (1:500, HPA014518 Sigma), anti-TREM2 (1:200, AF1828 R&D Systems), anti-CD68 (1:100, M0718 Dako), anti-TMEM119 (1:100, ab185333 Abcam), anti-IBA1 (1:500; Wako, 019-19741).

Filipello F., You S.F., Mirfakhar F.S., Mahali S., Bollman B., Acquarone M., Korvatska O., Marsh J.A., Sivaraman A., Martinez R., Cantoni C., De Feo L., Ghezzi L., Minaya M.A., Renganathan A., Cashikar A.G., Satoh J.I., Beatty W., Iyer A.K., Cella M., Raskind W.H., Piccio L, & Karch C.M. (2023). Defects in lysosomal function and lipid metabolism in human microglia harboring a TREM2 loss of function mutation. Acta Neuropathologica, 145(6), 749-772.

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Immunofluorescence Staining of Cultured Cells

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Cultured cells were washed once in PBS and fixed with 4% paraformaldehyde (10 min room temperature) followed by 3 PBS washes. Cells were permeabilized by treating with 100% ice‐cold EtOH for 2 min at room temperature followed by 2 × PBS washes. Fixed cells were incubated with blocking buffer (3% (v/v) goat/chicken serum, 0.1% (v/v) Triton‐X‐100 in PBS) for 1 h at room temperature before overnight incubation at 4oC with primary antibodies diluted in blocking buffer. Primary antibodies used were anti‐IBA‐1 (1:100 Abcam AB5076), anti‐Glut5 (1:100 R&D systems MAB1349), anti‐TMEM119 (1:100 Abcam AB185333) and anti‐P2RY12 (1:100 Abcam AB188968). Following overnight incubation, cells were three times with PBS prior to a 1‐h incubation at room temperature in the dark with fluorescent secondary antibodies diluted in blocking solution. Secondary antibodies used were Alexa Fluor 594 chicken anti‐goat IgG (Invitrogen A21468), Alexa Fluor 594 goat anti‐rabbit IgG (Invitrogen A11037) and Alexa Fluor 488 goat anti‐mouse IgG (Invitrogen A11029) all at 1:400 (Appendix Fig S8).
Coverslips were subsequently incubated with Hoechst 33258 (Thermo Fisher Scientific) at 1:5,000 in blocking buffer and mounted on microscope slides (Immu‐Mount, Fisher, 9990402). Images were taken using a Leica DM18 confocal microscope (Appendix Fig S8C).

Maguire E., Menzies G.E., Phillips T., Sasner M., Williams H.M., Czubala M.A., Evans N., Cope E.L., Sims R., Howell G.R., Lloyd‐Evans E., Williams J., Allen N.D, & Taylor P.R. (2021). PIP2 depletion and altered endocytosis caused by expression of Alzheimer's disease‐protective variant PLCγ2 R522. The EMBO Journal, 40(17), e105603.

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Immuno-histochemical Analysis of Tumor Tissue

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We dropped fresh tumor tissue in 4% paraformaldehyde overnight at 4 °C. After being washed with PBS three times for 10 min, the fixed tissue was placed in 20% and 30% sucrose solution to dehydrate and then was embedded in optimal cutting temperature medium (Thermo Scientific). Embedded tissue was sectioned at 20–30 μm cryosections using Leica CM3050S cryostat. Before IHC staining, tissue slices were subjected to heat-mediated antigen retrieval with Tris/EDTA buffer (PH 9.0). Then, tissue slices were blocked in 5% donkey serum for 2 h. Sections were incubated with primary antibody at the following dilutions: goat anti-IBA1 (1:100, Abcam, ab5076), Rabbit anti-MCP1 (1:100, Abcam, ab73680), mouse anti-CD44 (1:100, Abcam, ab16728), rabbit anti-VWF (1:100, Abcam, ab9378), mouse anti-CD3 (1:50, Abcam, ab699), rabbit anti-cleaved Caspase-3 (1:200, Cell Signaling, #9661), rabbit anti-TMEM119 (1:1000, Abcam, ab185333). After incubation overnight at 4 °C with primary antibody, Alexa Fluor 488, 594, or 647 fluorophore-conjugated secondary antibodies (1:500) (Life Technologies), and DAPI (1:500) were added to slices incubation buffer for 2 h. We used Olympus FV3000 confocal microscope for collecting images.

Zhang Q., Cheng S., Wang Y., Wang M., Lu Y., Wen Z., Ge Y., Ma Q., Chen Y., Zhang Y., Cao R., Li M., Liu W., Wang B., Wu Q., Jia W, & Wang X. (2021). Interrogation of the microenvironmental landscape in spinal ependymomas reveals dual functions of tumor-associated macrophages. Nature Communications, 12, 6867.

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FKBP51 Protein Localization in Brain

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To quantify cellular and subcellular localisation of FKBP51 protein, triple-label immunohistochemistry was performed in postmortem brain Series 3. Fresh frozen tissue blocks were cryosectioned at 50µm, mounted onto Superfrost Plus Gold slides. Sections were post-fixed with 4%
PFA and antigen retrieval was performed using 10mM citric acid buffer with 0.5% Tween-20 for 10mins. Tissue was permeabilised with 0.3% Triton X-100 in phosphate buffered saline (PBS) for 5mins and blocked with 10% normal serum, 1% bovine serum albumin, with 0.3M glycine in PBS at room temperature for 1hr. Tissue was then incubated overnight at 4°C in a primary antibody solution containing rabbit anti-FKBP51 (1:300, Cell Signalling, #8245S or 1:300, Santa Cruz Biotechnology, #D-4; antibodies validated: Figure S1), neuronal marker (1:300 mouse anti-NeuN, Millipore MAB377), microglia marker (1:100, rabbit anti-TMEM119, Abcam #ab185333) and astrocyte marker
(1:100, chicken anti-GFAP, Millipore AB5541). Following washing, appropriate secondary antibodies and DAPI were applied (anti-rabbit 488, anti-chicken 546, anti-mouse 647 at 1:150; DAPI at 1:5,000).
Tissues were counterstained with autofluorescence eliminator reagent (Merck Millipore-2160) and cover-slipped with Aqua-Poly/Mount medium. Images were captured on the Leica SP8 confocal microscope at 40x magnification.

Matosin N., Arloth J., Czamara D., Edmond K.Z., Maitra M., Fröhlich A.S., Martinelli S., Kaul D., Bartlett R., Curry A.R., Gassen N.C., Hafner K., Mueller N.S., Worf K., Rehawi G., Nagy C., Halldorsdottir T., Cruceanu C., Gagliardi M., Gerstner N., Ködel M., Murek V., Ziller M.J., Scarr E., Tao R., Jaffe A.E., Arzberger T., Falkai P., Kleinmann J.E., Weinberger D.R., Mechawar N., Schmitt A., Dean B., Turecki G., Hyde T.M., & Binder E.B. (2021). Associations of psychiatric disease and aging onFKBP5expression converge on cortical supragranular neurons.

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