All lanes : Anti-PU.1/Spi1 antibody [EPR22624-20] - ChIP Grade (ab227835) at 1/1000 dilution

Lane 1 : RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate
Lane 2 : NIH/3T3 (mouse embryonic fibroblast), whole cell lysate
Lane 3 : J774A.1 (mouse reticulum cell sarcoma monocyte macrophage), whole cell

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 31 kDa

Blocking and dilution buffer: 5% NFDM/TBST .

Exposure times.

Lanes 1-2: 26 seconds; Lane 3: 3 minuters.

Negative control: NIH/3T3 cell line (PMID: 23509362, 16331260).

Chromatin was prepared from J774A.1 cells according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with 1.5 mM EGS for 30 mins and then formaldehyde for 10 min.

The ChIP was performed with 25 µg of chromatin, 5 µg of ab227835 (red), or 5 µg of rabbit normal IgG ab172730 (gray) and 20 µl of Protein A/G sepharose beads. The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci).

Primers and probes are from paper PMID: 11869689

*http://www.abcam.com/resources?keywords=X%20ChIP%20protocol

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling Spi1 with ab227835 at 1/5000 dilution (0.103 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining in Kupffer cells of moue liver. The section was incubated with ab227835 for 15 mins at RT. The immunostaining staining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control/ Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling Spi1 with ab227835 at 1/5000 dilution (0.103 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on mouse spleen. The section was incubated with ab227835 for 15 mins at RT. The immunostaining staining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control/ Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Spi1 was immunoprecipitated from 0.35 mg J774A.1 (mouse reticulum cell sarcoma monocyte macrophage) whole cell lysate with ab227835 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab227835 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: J774A.1 (mouse reticulum cell sarcoma monocyte macrophage) whole cell lysate 10ug

Lane 2: ab227835 IP in J774A.1 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab227835 in J774A.1 whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 30 seconds

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cells labelling Spi1 with ab227835 at 1/50 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) isotype control (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Immunofluorescent analysis of 4% parafoprmal;dehyde-fixed, 0.1% Triton X-100 permeabilized RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) cells labeling PU.1/Spi1 with ab227835 at 1/500 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution (green). Confocal image showing nuclear staining in RAW 264.7 cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1/200 dilution (red).

Secodary antibody only control: Used PBS instead of primary antibody, followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 dilution.