Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of testes from juvenile (PND 7 and PND 14) mice labeling Sall4 with ab57577. Testes were dissected and fixed in 4% PFA overnight at 4°C. Fixed tissues were processed for paraffin embedding and 5 µm serial sections were collected. Sections were deparaffinized in xylene (2×15 min), rehydrated in a graded ethanol series (2×100% for 10 min, 1×95% for 5 min, 1×80% for 5 min, 1×70% for 5 min, 1×50% for 5 min, 1×25% for 5 min) and rinsed in 1× DPBS. Slides were then incubated in sodium citrate antigen retrieval buffer (10 mM Sodium Citrate, 0.05% Tween-20, pH 6) for 30 min at 97.5°C and allowed to cool to room temperature. After rinsing twice in 1× DPBS containing 0.1% Tween-20 (DBPS-T), unspecific binding sites in tissue sections were saturated by incubation with blocking buffer (1× DPBS containing 3% bovine serum albumin, 0.1% Triton X-100 and 5% normal serum from the host species of the secondary antibody) for 30 min at room temperature. Primary antibodies were diluted in blocking buffer and added to tissue sections for 90 min at room temperature.

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IHC-P image of Sall4 staining with ab57577 on tissue sections from adult mouse testis. The sections were subjected to heat-mediated antigen retrieval using Dako antigen retrieval solution. The sections were then blocked with 5% milk for 30 minutes at 25°C, before incubation with ab57577 (1/100 dilution) for 18 hours at 4°C. The secondary was an Alexa-Fluor 488 conjugated goat anti-mouse polyclonal, used at a 1/500 dilution.

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Sall4 antibody (ab57577) at 1ug/lane + HeLa cell lysate at 25ug/lane.

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Sall4 antibody (ab57577) used in immunohistochemistry at 5ug/ml on formalin fixed and paraffin embedded human testis.

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Overlay histogram showing HeLa cells stained with ab57577 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab57577, 1µg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde/permeabilized in 0.1% PBS-Tween used under the same conditions.

This image was generated using the ascites version of the product.

IHC-P image of Sall4 staining with ab57577 on tissue sections from adult marmoset testis. The sections were subjected to heat-mediated antigen retrieval using Dako antigen retrieval solution. The sections were then blocked with 5% milk for 30 minutes at 25°C, before incubation with ab57577 (1/100 dilution) for 18 hours at 4°C. The secondary was an Alexa-Fluor 488 conjugated goat anti-mouse polyclonal, used at a 1/500 dilution.

This image was generated using the ascites version of the product.

See Abreview