Anti-IL-33 antibody [EPR17831]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-IL-33 antibody [EPR17831] (AB187060)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) cells labeling IL33 with ab187060 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing increased nuclear staining in RAW 264.7 cells treated with 50 nM Phorbol-12-myristate-13-acetate (PMA) and 5 μg/ml Lipopolysaccharide for 24h.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IL-33 antibody [EPR17831] (AB187060)

Immunohistochemical analysis of paraffin-embedded rat spleen tissue labeling IL33 with ab187060 at 1/2000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101), ready to use. Nuclear staining in endothelial cells of rat spleen is observed (PMID : 12819012). Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101), ready to use.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-IL-33 antibody [EPR17831] (AB187060)

Intracellular Flow Cytometry analysis ofRAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 50nM PMA and 5μg/ml LPS for 24h (Red) / Untreated control (Green) labeling IL-33 with ab187060 at 1/500 dilution. Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 was used as the secondary antibody. Cells were fixed with4% paraformaldehyde and permeabilised with0.1% Tween-20.Isotype control - Rabbit monoclonal IgG (ab172730) (Black). Unlabelled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IL-33 antibody [EPR17831] (AB187060)

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling IL33 with ab187060 at 1/2000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101), ready to use. Nuclear staining in endothelial cells of mouse spleen is observed (PMID : 12819012). Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101), ready to use.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-IL-33 antibody [EPR17831] (AB187060)

Flow cytometry overlay histogram showing left, Raw264.7 treated with 50nM PMA and 5μg/ml LPS for 24h and right, negative untreated Raw264.7 stained with ab187060 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10μg/ml anti CD16/CD32 and 10% normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (ab187060) (1x 106 in 100μl at 1.0μg/ml (1/2090)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IL-33 antibody [EPR17831] (AB187060)

Immunohistochemical analysis of paraffin-embedded (A) Lymph node tissue from wild-type C57BL/6JGpt mice (B) Lymph node tissue from IL-33 knockout mice labeling IL33 with ab187060 at 1/5000 dilution, followed by LeicaDS9800 (Bond™ Polymer Refine Detection), ready to use. Counter stained with Hematoxylin. Antigen retrieval was heat mediated with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.  Positive staining on (A) Lymph node tissue from wild-type C57BL/6JGpt mice and no staining on (B) Lymph node tissue from IL-33 knockout mice. The section was incubated with ab187060 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. The tissue samples were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and IL33-KO homozygous mice (Strain ID : T052437).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IL-33 antibody [EPR17831] (AB187060)

Immunohistochemical analysis of paraffin-embedded (A) Spleen tissue from wild-type C57BL/6JGpt mice (B) Spleen tissue from IL-33 knockout mice labeling IL33 with ab187060 at 1/5000 dilution, followed by LeicaDS9800 (Bond™ Polymer Refine Detection), ready to use. Counter stained with Hematoxylin. Antigen retrieval was heat mediated with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins. Positive staining on (A) Spleen tissue from wild-type C57BL/6JGpt mice and no staining on (B) Spleen tissue from IL-33 knockout mice. The section was incubated with ab187060 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. The tissue samples were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and IL33-KO homozygous mice (Strain ID : T052437).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IL-33 antibody [EPR17831] (AB187060)

Immunohistochemical analysis of paraffin-embedded (A) Lung tissue from wild-type C57BL/6JGpt mice (B) Lung tissue from IL-33 knockout mice labeling IL33 with ab187060 at 1/5000 dilution, followed by LeicaDS9800 (Bond™ Polymer Refine Detection), ready to use. Counter stained with Hematoxylin. Antigen retrieval was heat mediated with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins. Positive staining on (A) Lung tissue from wild-type C57BL/6JGpt mice and no staining on (B) Lung tissue from IL-33 knockout mice. The section was incubated with ab187060 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. The tissue samples were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and IL33-KO homozygous mice (Strain ID : T052437).

• WB

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Western blot - Anti-IL-33 antibody [EPR17831] (AB187060)

Blocking/Dilution : 5% NFDM/TBST

Exposure : 3 minutes

IL-33 expression is induced by LPS treatment of PMA-differentiated RAW 264.7 cells (PMID 19559631; PMID 19933859).

All lanes:

Western blot - Anti-IL-33 antibody [EPR17831] (ab187060) at 1/1000 dilution

Lane 1:

RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 10 µg

Lane 2:

RAW 264.7 treated with 50 nM Phorbol-12-myristate-13-acetate (PMA) and 5 ug/ml lipopolysaccharide (LPS) for 24 hours, whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>)

Predicted band size: 30 kDa

Observed band size: 33 kDa

true

• WB

Unknown

Western blot - Anti-IL-33 antibody [EPR17831] (AB187060)

Blocking/Dilution : 5% NFDM/TBST

Exposure : 3 minutes

All lanes:

Western blot - Anti-IL-33 antibody [EPR17831] (ab187060) at 1/1000 dilution

Lane 1:

Rat lung tissue lysate at 20 µg

Lane 2:

Mouse lung tissue lysate at 10 µg

Secondary

Lane 1:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Lane 2:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 30 kDa

Observed band size: 30 kDa

true

• WB

Lab

Western blot - Anti-IL-33 antibody [EPR17831] (AB187060)

Loading control : Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1/200000) (36KDa) Blocking/Diluting buffer and concentration : 5% NFDM/TBST The tissue samples were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and IL33-KO homozygous mice (Strain ID : T052437).

Lanes 1 - 9:

Western blot - Anti-IL-33 antibody [EPR17831] (ab187060) at 1/1000 dilution

Lanes 1 - 9:

Western blot - Anti-IL-33 antibody [EPR17831] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/il-33-antibody-epr17831-bsa-and-azide-free-ab229698'>ab229698</a>)

Lane 1:

Wild-type mouse spleen tissue lysate (male)

Lanes 2 - 3:

IL-33 knockout spleen tissue lysate (male)

Lane 4:

Wild-type mouse inguinal lymph nodes tissue lysate (male)

Lanes 5 - 6:

IL-33 knockout inguinal lymph nodes tissue lysate (male)

Lane 7:

Wild-type mouse submandibular lymph nodes tissue lysate (male)

Lanes 8 - 9:

IL-33 knockout submandibular lymph nodes tissue lysate (male)

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 15-30 kDa

false

Exposure time: 38s

• WB

Lab

Western blot - Anti-IL-33 antibody [EPR17831] (AB187060)

Loading control : Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1/200000) (36KDa) Blocking/Diluting buffer and concentration : 5% NFDM/TBST The tissue samples were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and IL33-KO homozygous mice (Strain ID : T052437).

Lanes 1 - 3:

Western blot - Anti-IL-33 antibody [EPR17831] (ab187060)

Lanes 1 - 3:

Western blot - Anti-IL-33 antibody [EPR17831] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/il-33-antibody-epr17831-bsa-and-azide-free-ab229698'>ab229698</a>)

Lane 1:

Wild-type mouse lung tissue lysate (male)

Lanes 2 - 3:

IL-33 knockout lung tissue lysate (male)

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 15-30 kDa

false

Exposure time: 38s