Ito T, Kaku-Ito Y, Murata M, et al. used ab228461 at 1/100 dilution in immunohistochemical analysis of human primary cutaneous acral melanoma tissue. Antigen retrieval was performed using Heat Processor Solution pH 9 (Nichirei Biosciences, Tokyo, Japan) at 100 °C for 45 min. 

Matched pairs of primary and metastatic acral melanomas from three patients with discordant staining between primary and metastatic lesions. Black rectangles show representative high-power views of each figure (A,D,F). Bars indicate 2 mm in (A,B,D–F) and 200 μm in (C).

Ito T, Kaku-Ito Y, Murata M, et al. used ab228461 at 1/100 dilution in immunohistochemical analysis of human primary cutaneous acral melanoma tissue. Antigen retrieval was performed using Heat Processor Solution pH 9 (Nichirei Biosciences, Tokyo, Japan) at 100 °C for 45 min. 

Three cases of acral melanomas with homogeneous VE1-positive staining. (A) Strong (3+) VE1 signals are evident. (B) High-powered view of the rectangular area in A. (C) Moderate (2+) VE1 staining of acral melanoma. (D) High-powered view of the rectangular area in C. Some tumor-infiltrating macrophages showing a strong red color (arrows) are noted. These macrophages should be excluded from the assessment of VE1 staining in melanoma. (E) Weak (1+) but definitely positive staining of VE1. (F) High-powered view of the rectangular area in E. Lentiginous spread of melanoma cells in the basal layer (*) is evident. Ascending melanoma cells (arrows) in the epidermis as well as melanoma cells in the dermis are also VE1 positive. Bars indicate 500 μm in A, 200 μm in B,F, 1 mm in C,E, and 100 μm in D.

Ito T, Kaku-Ito Y, Murata M, et al. used ab228461 at 1/100 dilution in immunohistochemical analysis of human primary cutaneous acral melanoma tissue. Antigen retrieval was performed using Heat Processor Solution pH 9 (Nichirei Biosciences, Tokyo, Japan) at 100 °C for 45 min. 

A case of acral melanoma with heterogeneous VE1 staining. VE1-positive signals are shown in red. (A) VE1-positive red melanoma cells and VE1-negative melanoma cells are intermingled. (B) High-powered view of the rectangular area in A. Cells with brown pigments and strongly red cells are scattered. (C) CD68 staining of the same area as in A. Positive signals of CD68 are shown in brown. (D) High-powered view of the rectangular area in C. Brown cells in D are tumor-infiltrating macrophages. (E) Melan A staining of the same area as in A and C. Positive signals are shown in red. (F) High-powered view of the rectangular area in E. Melan A staining clearly shows melanoma cells. Bars indicate 500 μm in A,C,E, and 200 μm in B,D,F.

Ito T, Kaku-Ito Y, Murata M, et al. used ab228461 at 1/100 dilution in immunohistochemical analysis of human primary cutaneous acral melanoma tissue. Antigen retrieval was performed using Heat Processor Solution pH 9 (Nichirei Biosciences, Tokyo, Japan) at 100 °C for 45 min. 

A negative case of VE1 staining. Some cells show strong VE1 expression in red (A,B), but CD68 staining clearly highlights that these VE1-positive cells are macrophages (C,D). No melanoma cells stain with VE1 and this case should be regarded as “negative” for VE1. (B) High-powered view of the rectangular area in A. (D) High-powered view of the rectangular area in C. Bars indicate 1 mm in A,C and 200 μm in B,D.

Samples from human tumor tissues that were Positive (A, E, I) and Negative (B, F, J) for BRAF expression. Boxes in A, E, I show the negative controls from their corresponding non-tumor tissues. 

IHC for BRAF protein expression was performed on 4 μm-thick sections of formalin-fixed, paraffin-embedded tissues, using ab228461. The specimens were fixed in 10% neutral buffered formalin for 24–48 hours. after deparaffinization, the slides were pretreated with cell conditioning 1 for 64 minutes for antigen unmasking and followed by pre-primary antibody peroxidase inhibition. The slides were then incubated with the VE1 antibody at 37°C for 16 minutes, and counterstained with hematoxylin II for 4 minutes and bluing reagent for 4 minutes.

BRAF V600E IHC positive PXA cases molecularly confirmed as BRAF V600E mutant tumors: strong granular cytoplasmic immunostaining of a characteristic pleomorphic multinucleated giant tumor cell (A), weak granular cytoplasmic immunostaining of pleomorphic and spindle tumor cells (B), strong granular cytoplasmic immunostaining of a cluster of isolated tumor cells (C).

Four-micron freshly cut sections (

BRAF V600E mutations in adamantinomatous craniopharyngioma. Two cases are shown (a–e and f–j). a, f Classical features of aCP (wet keratin, stellate reticulum, palisaded epithelium). b, g Translocation of β-catenin is shown in both cases (brown reaction product). c, h An antibody to BRAF V600E (VE1) shows staining of the tumour tissue (brown reaction product).

Immunohistochemical analysis was performed on 4µm sections from FFPE tumour specimens. Following dewaxing through graded alcohols, endogenous peroxidase activity was blocked (3% (v/v) H2O2 in PBS, pH 7.3, 20 minutes with orbital shaking). Epitope retrieval was achieved by autoclaving in sodium citrate (10mM, pH 6.0, 10 minutes) (β-catenin) or Tris-EDTA (10mM Tris base, 1mM EDTA, pH 9.0, 10 minutes) (BRAF). Sections were blocked with serum block for 15 minutes, then incubated overnight at 4°C with primary antibody diluted in PBS or TBS-T (1:1000 in PBS (β-catenin); 1:50 in TBS-T (BRAF)).

Formalin-fixed, paraffin-embedded human colon carcinoma tissue stained for B Raf (mutated V600E) using ab228461 at a 1/100 dilution in immunohistochemical analysis.