Anti-DAZL antibody [EPR21028]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DAZL antibody [EPR21028] (AB215718)

Immunohistochemical analysis of paraffin-embedded human testis tissue labeling DAZL with ab215718 at 1/8000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining in primary spermatocytes of human testis (PMID : 24746554) is observed. Counterstained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DAZL antibody [EPR21028] (AB215718)

Immunohistochemical analysis of paraffin-embedded rat testis tissue labeling DAZL with ab215718 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining in spermatogonia and primary spermatocytes of rat testis (PMID : 24746554) is observed. Counterstained with hematoxylin

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-DAZL antibody [EPR21028] (AB215718)

Immunohistochemical analysis of 4% paraformaldehyde-fixed, 0.2% Triton-X-100 permeabilized frozen mouse testis tissue labeling DAZL with ab215718 at 1/300 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® staining in the pachytene spermatocytes and secondary spermatocytes, not present in Sertoli, Leydig or myoid cells (PMID : 24746554).

The nuclear counter stain is DAPI (blue).

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

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-DAZL antibody [EPR21028] (AB215718)

Immunohistochemical analysis of 4% paraformaldehyde-fixed, 0.2% Triton-X-100 permeabilized frozen rat testis tissue labeling DAZL with ab215718 at 1/60 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Cytoplasmic staining in the pachytene spermatocytes and secondary spermatocytes, not present in Sertoli, Leydig or myoid cells (PMID : 24746554).

The nuclear counter stain is DAPI (blue).

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

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-DAZL antibody [EPR21028] (AB215718)

ab215718 staining DAZL in mES cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab215718 at 1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue). Also suitable in cells fixed with 100% methanol (5 min). Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DAZL antibody [EPR21028] (AB215718)

Immunohistochemical analysis of paraffin-embedded mouse testis tissue labeling DAZL with ab215718 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining in spermatogonia and primary spermatocytes of mouse testis (PMID : 24746554) is observed. Counterstained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IP

Supplier Data

Immunoprecipitation - Anti-DAZL antibody [EPR21028] (AB215718)

DAZL was immunoprecipitated from 0.35 mg mouse testis lysate with ab215718 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab215718 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

Lane 1 : Mouse testis lysate 10 μg (Input).

Lane 2 : ab215718 IP in mouse testis lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab215718 in mouse testis lysate.

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

Exposure time : 3 seconds.

All lanes:

Immunoprecipitation - Anti-DAZL antibody [EPR21028] (ab215718)

Predicted band size: 33 kDa

false

• WB

Supplier Data

Western blot - Anti-DAZL antibody [EPR21028] (AB215718)

Blocking/Dilution buffer : 5% NFDM/TBST.

The blot was developed on a BIO-RAD^® ChemiDoc™ MP instrument.

All lanes:

Western blot - Anti-DAZL antibody [EPR21028] (ab215718) at 1/1000 dilution

Lane 1:

Mouse testis lysate at 20 µg

Lane 2:

Rat testis lysate at 20 µg

Lane 3:

Human testis lysate at 20 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>) at 1/100000 dilution

Predicted band size: 33 kDa

Observed band size: 38 kDa

true

Exposure time: 3min

• WB

AbReview66346****

Western blot - Anti-DAZL antibody [EPR21028] (AB215718)

10% gel.

Blocked with 5% milk for 1 hour at room temperature.

Incubated with the primary antibody for 12 hours at 4°C in TBST.

All lanes:

Western blot - Anti-DAZL antibody [EPR21028] (ab215718) at 1/1000 dilution

Lane 1:

Mouse testis tissue lysate at 25 µg

Lane 2:

DAZL knockout mouse testis tissue lysate at 25 µg

Secondary

All lanes:

HRP-conjugated horse anti-rabbit IgG at 1/10000 dilution

Predicted band size: 33 kDa

Observed band size: 37 kDa

true

Exposure time: 3s

This image is courtesy of an anonymous collaborator abreview.

• WB

CiteAb

Western blot - Anti-DAZL antibody [EPR21028] (AB215718)

DAZL western blot using anti-DAZL antibody [EPR21028] ab215718. Publication image and figure legend from Yang, C. R., Rajkovic, G., et al., 2020, Nat Commun, PubMed 32170089.

ab215718 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab215718 please see the product overview.

Interference with Dazl mRNA translation depletes oocytes of the DAZL protein and inhibits translation of a specific downstream target.a DAZL protein accumulation during the transition from the GV-to-MI stages of oocyte maturation. GV stage oocytes from wild type mice were cultured in vitro up to 8 h of maturation and used for Western blot analysis. 150 oocytes per lane was loaded on the gel and accumulation of α-tubulin was used as a loading control. The complete time course was performed once, while changes between the first and last time point confirmed in two additional independent experiments (see Supplementary Fig 1a). b Morpholino oligonucleotide (MO) down-regulation of DAZL protein. GV stage oocytes from RiboTagfl/fl : Zp3-Cre : WT or RiboTagfl/fl : Zp3-Cre : DAZL+/− mice were injected with CON-MO or DAZL-MO as described in the methods. After 6 h, oocytes were collected and used for Western blot analysis. A representative experiment of the three performed is reported. c–e GV stage oocytes from RiboTagfl/fl : Zp3-Cre or RiboTagfl/fl : Zp3-Cre : Dazl+/− mice were injected with CON-MO or DAZL-MO and preincubated overnight in 2 µM milrinone, then cultured in inhibitor-free medium for maturation. Oocytes were collected at 0 and 6 h for RiboTag IP followed by qPCR analysis. Ribosome loading of the endogenous Dazl mRNA (unpaired two tailed t-test, p = 0.0022) and Tex19.1 (unpaired two tailed t-test, p < 0.0001), but not CcnB1 (unpaired two tailed t-test, P = 0.8567) mRNA is disrupted after DAZL depletion. (GV : germinal vesicle; MI : Meiosis I. Each dot represents an independent biological sample collected from different experiments done in different days. f–h Input data for the immunoprecipitation reported in c–e. When N > 2, data are presented as Mean ± SEM and each dot represents an independent biological observation. Statistical significance among the different samples was evaluated with the Brown-Forsythe ANOVA test, assuming unequal SD. Calculated P values : Dazl = 0.6680, Tex19.1 = 0.9713, CcnB1 = 0.5404.

false