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93195
Microglia Neurodegeneration Module Antibody Sampler Kit
Primary Antibodies

Microglia Neurodegeneration Module Antibody Sampler Kit #93195

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Flow cytometric analysis of Raw264.7 cells (blue) and J774A.1 cells (green) using ASC/TMS1 (D2W8U) Rabbit mAb (Mouse Specific) (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

Flow cytometric analysis of NIH/3T3 cells (red) and 32D cells (blue), using HS1 (D5A9) XP® Rabbit mAb (Rodent Specific).

Flow cytometric analysis of Daudi cells (blue) and U266 cells (green) using Cathepsin B (D1C7Y) XP® Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

Chromatin immunoprecipitations were performed with cross-linked chromatin from MCF7 cells treated with cobalt chloride (100 μM) overnight and HIF-1α (D1S7W) XP® Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across ARRDC3, a known target gene of HIF-1α (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product data sheet.

Confocal immunofluorescent analysis of HeLa cells, treated with either 10 μM MG132 (left) or 10 μM MG132 and 1 mM DMOG (right), using Hydroxy-HIF-1α (Pro564) (D43B5) XP® Rabbit mAb (green). Actin filaments have been labeled using DY-554 phalloidin (red).

Confocal immunofluorescent analysis of SK-MEL-28 (left) and LNCaP (right) cells, using Galectin-3/LGALS3 (D4I2R) XP® Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Flow cytometric analysis of Jurkat cells (blue) and DU145 cells (green) using Axl (C89E7H4) Rabbit mAb.

Flow cytometric analysis of Jurkat cells (blue) and THP-1 cells (green) using CD68 (D4B9C) XP® Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

Immunohistochemical analysis of paraffin-embedded human tonsil using CD68 (D4B9C) XP® Rabbit mAb.

Immunoprecipitation of CD68 from U-937 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is CD68 MultiMab™ Rabbit mAb mix. Western blot analysis was performed using CD68 MultiMab™ Rabbit mAb mix.

Confocal immunofluorescent analysis of mouse Tg2576 brain which overexpresses mutant human APP695. Sections were first labeled with HS1 (D5A9) XP® Rabbit mAb (Rodent Specific) (green) and APP/β-Amyloid (NAB228) Mouse mAb #2450 (yellow). After blocking free secondary binding sites with Mouse (G3A1) mAb IgG1 Isotype Control #5415, sections were incubated with GFAP (GA5) Mouse mAb (Alexa Fluor® 647 Conjugate) #3657 (red). Nuclei were labeled with Hoechst 33342 #4082 (blue).

Confocal immunofluorescent analysis of Malme-3M (left) and MCF7 (right) cells using Cathepsin (D1C7Y) XP® Rabbit mAb (green) and β-Actin (8H10D10) Mouse mAb #3700 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Chromatin immunoprecipitations were performed with cross-linked chromatin from MCF7 cells treated with cobalt chloride (100 μM, overnight) and either HIF-1α (D1S7W) XP® Rabbit mAb or Normal Rabbit IgG #2729, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. The enriched DNA was quantified by real-time PCR using human ARRDC3 downstream primers, SimpleChIP® Human ERRFI1 Upstream Primers #31180, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

Western blot analysis of extracts from HeLa cells, treated with either 10 μM of MG132 (to accumulate hydroxylated HIF-1α) or 10 µM MG132 and 1 mM DMOG (to accumulate nonhyroxylated HIF-1α), using Hydroxy-HIF-1α (Pro564) (D43B5) XP® Rabbit mAb (upper) or total HIF-1α Antibody #3716 (lower).

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Galectin-3/LGALS3 (D4I2R) XP® Rabbit mAb.

Confocal immunofluorescent analysis of DU 145 (left) and HCC827 (right) cells using Axl (C89E7) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Flow cytometric analysis of live human peripheral blood mononuclear cells co-stained with anti-human CD14 using CD68 (D4B9C) XP® Rabbit mAb (right) compared to a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (left). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

Immunohistochemical analysis of paraffin-embedded human serous papillary carcinoma of the ovary using CD68 (D4B9C) XP® Rabbit mAb.

Western blot analysis of extracts from THP-1, U-937, and Jurkat cells using CD68 MultiMab™ Rabbit mAb mix (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Confocal immunofluorescent analysis of mouse primary bone marrow-derived macrophages (BMDMs) either untreated (upper left) or treated with LPS (50 ng/ml, 4 hr, middle) or LPS followed by ATP (5 mM, 45 min, upper right), and J774A.1 (lower left) or Raw 264.7 (lower right) cells, using ASC/TMS1 (D2W8U) Rabbit mAb (Mouse Specific) (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye). Note the translocation of ASC to inflammasomes following stimulation with LPS and ATP (white arrows).

Confocal immunofluorescent analysis of 32D cells (left) and C2C12 cells (right), using HS1 (D5A9) XP® Rabbit mAb (Rodent Specific) (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Cathepsin B (D1C7Y) XP(R) Rabbit mAb.

Flow cytometric analysis of U-2 OS cells, untreated (blue) or treated with DMOG (1 mM, 6 h; green), using HIF-1α (D1S7W) XP® Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

Immunohistochemical analysis of paraffin-embedded SK-MEL-28 (left) and LNCaP (right) cell pellets using Galectin-3/LGALS3 (D4I2R) XP® Rabbit mAb.

Immunohistochemical analysis of paraffin-embedded breast carcinoma using Axl (C89E7) Rabbit mAb. Note staining of infiltrating cells.

Flow cytometric analysis of fixed and permeabilized human peripheral blood mononuclear cells co-stained with anti-human CD14 using CD68 (D4B9C) XP® Rabbit mAb (right) compared to a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (left). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using CD68 (D4B9C) XP® Rabbit mAb.

Immunoprecipitation of ASC/TMS1 from J774A.1 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is ASC (D2W8U) Rabbit mAb (Mouse Specific). Western blot analysis was performed using ASC/TMS1 (D2W8U) Rabbit mAb (Mouse Specific).

Immunohistochemical analysis of paraffin-embedded mouse spleen using HS1 (D5A9) XP® Rabbit mAb (Rodent Specific).

Immunohistochemical analysis of paraffin-embedded normal human kidney using Cathepsin B (D1C7Y) XP(R) Rabbit mAb.

Confocal immunofluorescent analysis of Hep G2 cells, untreated (left) or treated with cobalt chloride (500 μM, 24 h; right), using HIF-1α (D1S7W) XP® Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red).

Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma using Galectin-3/LGALS3 (D4I2R) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).

Immunohistochemical analysis of paraffin-embedded human renal cell carcinoma using Axl (C89E7) Rabbit mAb.

Confocal immunofluorescent analysis of THP-1 (left) and Jurkat (right) cells using CD68 (D4B9C) XP® Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye). Projected images from a z-stack series are shown.

Immunohistochemical analysis of paraffin-embedded THP-1 (left) and Jurkat (right) cell pellets using CD68 (D4B9C) XP® Rabbit mAb.

Western blot analysis of extracts from J774A.1 and Raw 264.7 cells using ASC/TMS1 (D2W8U) Rabbit mAb (Mouse Specific) (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Immunohistochemical analysis of paraffin-embedded LL2 syngeneic tumor using HS1 (D5A9) XP® Rabbit mAb (Rodent Specific).

Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma using Cathepsin B (D1C7Y) XP(R) Rabbit mAb.

Immunoprecipitation of HIF-1α from lysate of Hep G2 cells treated with cobalt chloride (100 µM, 4 h). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is HIF-1α (D1S7W) XP® Rabbit mAb. Western blot analysis was performed using HIF-1α (D1S7W) XP® Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as the secondary antibody.

Immunoprecipitation of Galectin-3/LGALS3 from SK-MEL-28 cell extracts. Lane 1 is 10% input, lane 2 is Normal Rabbit IgG #2729, and lane 3 is Galectin-3/LGALS3 (D4I2R) XP® Rabbit mAb (lane 3).

Immunohistochemical analysis of paraffin-embedded human B-cell non-Hodgkin's lymphoma using Axl (C89E7) Rabbit mAb.

Multiplex immunohistochemical analysis of paraffin-embedded human lung adenocarcinoma using CD68 (D4B9C) XP® rabbit mAb (red), PD-1 (EH33) mouse mAb #43248 (green), CD8α (C8/144B) mouse mAb #70306 (magenta), Pan-keratin (C11) mouse mAb #4545 (cyan), LAG3 (D2G4O™) XP® rabbit mAb #15372 (orange), and TIM-3 (D5D5R™) XP® rabbit mAb #45208 (yellow).

Western blot analysis of cell extracts from Baf3, 32D, and mouse spleen using HS1 (D5A9) XP® Rabbit mAb (Rodent Specific).

Western blot analysis of extracts from Hep G2 cells untreated (-) or treated with cobalt chloride (100 µM, 4 h; +), Raji cells untreated (-) or treated with cobalt chloride (100 µM, 4 h; +) and U-2 OS cells untreated (-) or treated with DMOG (1 mM, 6 h; +) using HIF-1α (D1S7W) XP® Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Western blot analysis of extracts from various cell lines using Galectin-3/LGALS3 (D4I2R) XP® Rabbit mAb.

Immunohistochemical analysis of paraffin-embedded human ovarian serous carcinoma using Axl (C89E7) Rabbit mAb.

Multiplex immunohistochemical analysis of paraffin-embedded human ovarian carcinoma using CD68 (D4B9C) XP® rabbit mAb (orange), PD-L1 (E1L3N®) XP® rabbit mAb #13684 (red), PD-L2 (D7U8C™) XP® rabbit mAb (magenta) #82723, Arginase-1 (D4E3M™) XP® rabbit mAb #93668 (green), IDO (D5J4E™) rabbit mAb #86630 (yellow), and Pan-keratin (C11) mouse mAb #4545 (cyan).

Western blot analysis of extracts from various cell lines using Cathepsin B (D1C7Y) XP® Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Immunohistochemical analysis of paraffin-embedded metastatic lung carcinoma using Axl (C89E7) Rabbit mAb.

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expressing full-length human Cathepsin B (hCTSB; +) or mouse Cathespin B (mCTSB; +) using Cathepsin B (D1C7Y) XP® Rabbit mAb.

Immunohistochemical analysis of paraffin-embedded cell pellets, NCI-H1299 (left) or Jurkat (right), using Axl (C89E7) Rabbit mAb.

Western blot analysis of extracts from various cell lines using Axl (C89E7) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).

To Purchase # 93195T
Product # Size Price
93195T
1 Kit  (8 x 20 µl) $ 538

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
ASC/TMS1 (D2W8U) Rabbit mAb (Mouse Specific) 67824 20 µl
  • WB
  • IP
  • IF
  • F
M 22 Rabbit IgG
HS1 (D5A9) XP® Rabbit mAb (Rodent Specific) 3892 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
M R 80 Rabbit IgG
Cathepsin B (D1C7Y) XP® Rabbit mAb 31718 20 µl
  • WB
  • IHC
  • IF
  • F
H M R 44, 27, 24 Rabbit IgG
HIF-1α (D1S7W) XP® Rabbit mAb 36169 20 µl
  • WB
  • IP
  • IF
  • F
  • ChIP
H M Mk 120 Rabbit IgG
Hydroxy-HIF-1α (Pro564) (D43B5) XP® Rabbit mAb 3434 20 µl
  • WB
  • IP
  • IF
H Mk 120 Rabbit IgG
Galectin-3/LGALS3 (D4I2R) XP® Rabbit mAb 87985 20 µl
  • WB
  • IP
  • IHC
  • IF
H 28 Rabbit IgG
Axl (C89E7) Rabbit mAb 8661 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
H Mk 138 Rabbit IgG
CD68 (D4B9C) XP® Rabbit mAb 76437 20 µl
  • IHC
  • IF
  • F
H Rabbit IgG
CD68 MultiMab™ Rabbit mAb mix 86985 20 µl
  • WB
  • IP
H 70-80, 130-140 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

Product Description

The Microglia Neurodegeneration Module Antibody Sampler Kit provides an economical means of detecting proteins identified as markers of microglial activity during neurodegeneration by western blot and/or immunofluorescence.

Specificity / Sensitivity

Each antibody in the Microglia Neurodegeneration Module Antibody Sampler Kit detects endogenous levels of its target protein. HS1 (D5A9) XP® Rabbit mAb (Rodent Specific) does not recognize human HS1 protein. HS1 has a calculated size of 54 kDa, but has an apparent molecular weight of 80 kDa on SDS-PAGE gels. Cathepsin B (D1C7Y) XP® Rabbit mAb recognizes endogenous levels of total cathepsin B protein and detects the heavy chain subunit of cathepsin B. Hydroxy-HIF-1α (Pro564) (D43B5) XP® Rabbit mAb detects endogenous levels of HIF-1α only when hydroxylated at Pro564 and may cross-react with other overexpressed proline hydroxylated proteins. Axl (C89E7) Rabbit mAb does not cross-react with Tyro3.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Leu310 of mouse HS1, Leu478 of human HIF-1α, the amino terminus of human Galectin-3/LGALS3, a hydroxypeptide surrounding Pro564 of human HIF-1α, and recombinant proteins specific to mouse ASC/TMS1, human Axl, human CD68, and the heavy chain subunit of human cathepsin B protein.

MultiMab™ rabbit monoclonal mix antibodies are prepared by combining individual rabbit monoclonal clones in optimized ratios for the approved applications. This product is optimized to detect CD68 as a monomer and a dimer by western blot and was produced by immunizing animals with recombinant human CD68 protein.

Background

Distinct microglial activation states have been identified using RNA-seq data from a vast array of neurological disease and aging models. These activation states have been categorized into modules corresponding to proliferation, neurodegeneration, interferon-relation, LPS-relation, and many others (1). Previous work identifying markers of specific brain cell types using RNA-seq has shown HS1 and ASC/TMS1 to be useful and specific tools to study microglia (2). HS1 is a protein kinase substrate that is expressed only in tissues and cells of hematopoietic origin (3) and ASC/TMS1 has been found to be a critical component of inflammatory signaling where it associates with and activates caspase-1 in response to pro-inflammatory signals (4).

CD68 is a common marker for macrophage lineage cells; with expression found in the lysosome making it a useful marker for activated phagocytic microglia (5). Galectin-3 has been shown to regulate inflammatory response in neurodegenerative diseases, released by microglia in response to inflammatory stimuli (6). Cathepsin B is a widely expressed cysteine peptidase located in the lysosome as well as processed and secreted, playing a role in microglial-mediated neuronal death (7). Hypoxia inducible factor-1 (HIF-1α) is a transcription factor responsible for adaptation to low oxygen environments whose downstream effects have been shown in a number of neurodegenerative diseases. Under normoxic conditions, HIF-1α is proline hydroxylated leading to ubiquitin mediated degradation (8). Axl is a receptor tyrosine kinase that binds Gas6, stimulating regulatory effects on microglial phagocytic response to inflammatory stimuli (9).

  1. Friedman, B.A. et al. (2018) Cell Rep 22, 832-47.
  2. Zhang, Y. et al. (2014) J Neurosci 34, 11929-47.
  3. Kitamura, D. et al. (1995) Biochem Biophys Res Commun 208, 1137-46.
  4. Srinivasula, S.M. et al. (2002) J Biol Chem 277, 21119-22.
  5. Hopperton, K.E. et al. (2018) Mol Psychiatry 23, 177-98.
  6. Yip, P.K. et al. (2017) Sci Rep 7, 41689.
  7. Gan, L. et al. (2004) J Biol Chem 279, 5565-72.
  8. Zhang, Z. et al. (2011) Curr Med Chem 18, 4335-43.
  9. Grommes, C. et al. (2008) J Neuroimmune Pharmacol 3, 130-40.

Pathways & Proteins

Explore pathways + proteins related to this product.

For Research Use Only. Not For Use In Diagnostic Procedures.

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