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83163
Microglia Cross Module Antibody Sampler Kit
Primary Antibodies

Microglia Cross Module Antibody Sampler Kit #83163

Western Blotting Image 1

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).

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Western Blotting Image 2

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

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Flow Cytometry Image 3

Flow cytometric analysis of Jurkat cells using Ki-67 (D3B5) Rabbit mAb and Propidium Iodide (PI)/RNase Staining Solution #4087. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

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Western Blotting Image 4

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

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IF-IC Image 5

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).

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Western Blotting Image 6

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).

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Chromatin IP-seq Image 7

Chromatin immunoprecipitations were performed with cross-linked chromatin from U266 cells treated with Human Interferon-α (IFN-α) #9906 (10nM) for 30 min and Stat2 (D9J7L) 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 USP18, a known target gene of Stat2 (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product data sheet.

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Western Blotting Image 8

Western blot analysis of extracts from various cell lines using Stat2 (D9J7L) Rabbit mAb. KARPAS cell line source: Dr Abraham Karpas at the University of Cambridge.

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Western Blotting Image 9

Western blot analysis of extracts from serum-starved U266 and A-431 cells, untreated (-) or treated with Human Interferon-α1 (hIFN-α1) #8927 (10 ng/ml; +) using Phospho-Stat2 (Tyr690) (D3P2P) Rabbit mAb (upper) and total Stat2 (D9J7L) Rabbit mAb #72604 (lower).

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Western Blotting Image 10

Western blot analysis of extracts from various cell lines using Lamin A/C (4C11) Mouse mAb.

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Western Blotting Image 11

Western blot analysis of extracts from various cell lines using IQGAP1 (D8K4X) XP® Rabbit mAb.

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Western Blotting Image 12

After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.

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IP Image 13

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).

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IHC-P (paraffin) Image 14

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

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IF-IC Image 15

Confocal immunofluorescent analysis of HeLa cells using Ki-67 (D3B5) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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IHC-P (paraffin) Image 16

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

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Chromatin IP Image 17

Chromatin immunoprecipitations were performed with cross-linked chromatin from U266 cells treated with Human Interferon-α (IFN-α) #9906 (100 ng/ml) for 30 min, and either Stat2 (D9J7L) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using human USP18 promoter primers, SimpleChIP® Human WARS Intron 1 Primers #30101, 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.

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IP Image 18

Immunoprecipitation of Stat2 from KARPAS-299 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Stat2 (D9J7L) Rabbit mAb. Western blot was performed using Stat2 (D9J7L) Rabbit mAb. KARPAS cell line source: Dr Abraham Karpas at the University of Cambridge.

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Flow Cytometry Image 19

Flow cytometric analysis of U266 cells, untreated (blue) or treated with IFN-α (green) using Phospho-Stat2 (Tyr690) (D3P2P) Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 fragment (Alexa Fluor 488 Conjugate) #4412 was used as a secondary antibody.

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Western Blotting Image 20

Western blot analysis of extracts from THP-1 cells, untreated or treated with cycloheximide (CHX, 10 μg/ml, overnight) followed by TNF-α #8902 (20 ng/ml, 4 hr), using Lamin A/C (4C11) Mouse mAb.

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IHC-P (paraffin) Image 21

breast:

Immunohistochemical analysis of paraffin-embedded human infiltrating papillary carcinoma of the breast using IQGAP1 (D8K4X) XP® Rabbit mAb.

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Flow Cytometry Image 22

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.

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IHC-P (paraffin) Image 23

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

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IF-F Image 24

Confocal immunofluorescent analysis of the ventricular zone in P21 mouse brain using Ki-67 (D3B5) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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IHC-P (paraffin) Image 25

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

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Flow Cytometry Image 26

Flow cytometric analysis of U266 cells using Stat2 (D9J7L) Rabbit mAb (blue) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (red). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor 647 Conjugate) was used as a secondary antibody.

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IF-IC Image 27

Confocal immunofluorescent analysis of A-431 cells, serum starved (left) or treated with IFNα (1000 U/ml for 30 min; right) using Phospho-Stat2 (Tyr690) (D3P2P) Rabbit mAb (green) and β-actin (8H10D10) Mouse mAb #3700 (red).

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IHC-P (paraffin) Image 28

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Lamin A/C (4C11) Mouse mAb.

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IHC-P (paraffin) Image 29

Immunohistochemical analysis of paraffin-embedded SK-MEL-28 (left) and LNCaP (right) cell pellets using IQGAP1 (D8K4X) XP® Rabbit mAb.

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IF-IC Image 30

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).

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Flow Cytometry Image 31

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

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IHC-P (paraffin) Image 32

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

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IF-IC Image 33

Confocal immunofluorescent analysis of A-431 cells, serum starved (left) or treated with IFNα (1000 U/ml for 30 mins; right) using Stat2 (D9J7L) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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IHC-P (paraffin) Image 34

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Lamin A/C (4C11) Mouse mAb.

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IHC-P (paraffin) Image 35

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using IQGAP1 (D8K4X) XP® Rabbit mAb.

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IF-IC Image 36

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).

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Flow Cytometry Image 37

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

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Flow Cytometry Image 38

Flow cytometric analysis of HeLa cells (green) using Lamin A/C (4C11) Mouse mAb (solid lines) or a concentration matched Mouse (G3A1) mAb IgG Isotype Control #5415 (dashed lines). Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4408 was used as a secondary antibody.

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IHC-P (paraffin) Image 39

Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma using IQGAP1 (D8K4X) XP® Rabbit mAb.

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IF-F Image 40

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).

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IF-IC Image 41

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

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IF-IC Image 42

Confocal immunofluorescent analysis of HeLa cells using Lamin A/C (4C11) Mouse mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).

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IF-IC Image 43

Confocal immunofluorescent analysis of A549 (left) and Hep G2 (right) cells using IQGAP1 (D8K4X) XP® Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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IF-F Image 44

Immunofluorescent analysis of normal rat brain using Lamin A/C (4C11) Mouse mAb (green) and MAP2 Antibody #4542 (red).

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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
Ki-67 (D3B5) Rabbit mAb 9129 20 µl
  • IF
  • F
H M R 359 Rabbit IgG
Axl (C89E7) Rabbit mAb 8661 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
H Mk 138 Rabbit IgG
Hydroxy-HIF-1α (Pro564) (D43B5) XP® Rabbit mAb 3434 20 µl
  • WB
  • IP
  • IF
H Mk 120 Rabbit IgG
Stat2 (D9J7L) Rabbit mAb 72604 20 µl
  • WB
  • IP
  • IF
  • F
  • ChIP
H M 97, 113 Rabbit IgG
Phospho-Stat2 (Tyr690) (D3P2P) Rabbit mAb 88410 20 µl
  • WB
  • IF
  • F
H R 97, 113 Rabbit IgG
Lamin A/C (4C11) Mouse mAb 4777 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
H M R Mk 74 (Lamin A), 63 (Lamin C) Mouse IgG2a
IQGAP1 (D8K4X) XP® Rabbit mAb 20648 20 µl
  • WB
  • IHC
  • IF
H M R Mk 195 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

The Microglia Cross Module Antibody Sampler Kit provides an economical means of detecting proteins identified as markers of microglial activity corresponding to proliferation, neurodegeneration, interferon and LPS-relation by western blot and/or immunofluorescence.

Each antibody in the Microglia Cross Module Antibody Sampler Kit detects endogenous levels of its target protein. Hydroxy-HIF-1α (Pro564) (D43B5) XP® Rabbit mAb detects endogenous levels of HIF-1α only when hydroxylated at Pro564. This antibody may cross-react with other overexpressed proline hydroxylated proteins. Phospho-Stat2 (Tyr690) (D3P2P) Rabbit mAb recognizes endogenous levels of Stat2 protein only when phosphorylated at Tyr690. Axl (C89E7) Rabbit mAb does not cross-react with Tyro3. 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. Lamin A/C (4C11) Mouse mAb detects endogenous levels of lamin A and lamin C proteins. It also reacts with the larger fragments of lamin A (50 kDa) and lamin C (41 kDa) produced by caspase cleavage during apoptosis. This antibody does not cross-react with lamins B1 and B2.

Monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Leu310 of mouse HS1, Pro564 of human HIF-1α, Leu706 of human Stat2, Tyr690 of human Stat2, the amino terminus of human Ki-67 and IQGAP1, and recombinant fragment of human Axl, human lamin A, and mouse ASC/TMS1.

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).

Ki-67 is a nuclear nonhistone protein (5) universally expressed among proliferating cells and absent in quiescent cells (6). Axl is a receptor tyrosine kinase that binds Gas6, stimulating regulatory effects on microglial phagocytic response to inflammatory stimuli (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). Stat2 is critical to the transcriptional responses induced by type I interferons, IFN-alpha/beta (9,10). In response to IFN-alpha/beta, Stat2 is activated by phosphorylation at site Tyr690 through associations with receptor-bound Jak kinases (11). Lamins are nuclear membrane structural components important for maintaining normal cell functions. Lamin A/C is cleaved by caspase-6 and serves as a marker for caspase-6 activation. The cleavage of lamins results in nuclear dysregulation and cell death (12,13). IQGAP1 is ubiquitously expressed and has been found to interact with APC (14) and the CLIP170 complex in response to small GTPases, promoting cell polarization and migration (15).

  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. Grommes, C. et al. (2008) J Neuroimmune Pharmacol 3, 130-40.
  6. Zhang, Z. et al. (2011) Curr Med Chem 18, 4335-43.
  7. Gerdes, J. et al. (1983) Int J Cancer 31, 13-20.
  8. Fu, X.Y. et al. (1992) Proc Natl Acad Sci U S A 89, 7840-3.
  9. Ihle, J.N. (2001) Curr Opin Cell Biol 13, 211-7.
  10. Weigel, M.T. and Dowsett, M. (2010) Endocr Relat Cancer 17, R245-62.
  11. Improta, T. et al. (1994) Proc Natl Acad Sci U S A 91, 4776-80.
  12. Watanabe, T. et al. (2004) Dev Cell 7, 871-83.
  13. Fukata, M. et al. (2002) Cell 109, 873-85.
  14. Oberhammer, F.A. et al. (1994) J Cell Biol 126, 827-37.
  15. Rao, L. et al. (1996) J Cell Biol 135, 1441-55.
Entrez-Gene Id
66824 , 558 , 3091 , 3059 , 8826 , 4288 , 4000 , 6773
Swiss-Prot Acc.
Q9EPB4 , P30530 , Q16665 , P14317 , P46940 , P46013 , P02545 , P52630
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.

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