Comparison of bleaching protocols utilizing hematoxylin and eosin stain and immunohistochemical proliferation marker MCM3 in pigmented melanomas

Jordan, Tierney; Williams, Destinee; Criswell, Sheila; Wang, Yongfu

doi:10.1080/01478885.2019.1649886

Cited by 11 publications

(14 citation statements)

References 9 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A recent study demonstrated that a complete bleaching of human melanoma samples can be reached placing the sections in a 3% H 2 O 2 solution containing 1% formamide and irradiating with a 60 W incandescent light bulb. 10 However, in this study, no control sections dipped in the same solution but not irradiated were used. Moreover, although the experiment was performed at RT, the temperature reached by the samples, due to both directly released heat and energy irradiated by the incandescent light bulb, has not been evaluated.…”

Section: Discussionmentioning

confidence: 99%

“…11 For these reasons and since a heated 3% H 2 O 2 solution can determine depigmentation of melanoma histological sections, it cannot be ruled out that the samples tested in that study benefited of a combined effect of H2O2 and heating. 10 In our study, particular attention was taken in maintaining previously-treated, intreatment and control sections in the same medium and at the same temperature, in order to avoid possible misleading effects deriving from variations of these parameters. Furthermore, the evaluation of the light source emission spectrum, allowed us to exclude a possible effect produced by infrared and ultraviolet radiations.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Bleaching melanin in formalin-fixed and paraffin-embedded melanoma specimens using visible light: a pilot study

et al. 2019

View full text Add to dashboard Cite

In fluorescence microscopy, light radiation can be used to bleach fluorescent molecules in formalin-fixed and paraffin-embedded (FFPE) samples, in order to increase the ratio between signal of interest and background autofluorescence. We tested if the same principle can be exploited in bright field microscopy to bleach pigmented melanoma FFPE sections together with cell morphology maintenance. After dewaxing and rehydration, serial FFPE sections of a feline diffuse iris melanoma, a canine dermal melanoma, a gray horse dermal melanoma and a swine cutaneous melanoma were irradiated with visible light for 1, 2, 3, 4 and 5 days, prior to Hematoxylin & Eosin staining. Complete bleaching was obtained after 1-day treatment in feline and swine melanomas, while 2 and 3 days were required in canine and equine neoplasms, respectively. In all treated samples, cell morphology was maintained. Photo-induced bleaching combined with immunohistochemistry was tested after a 3-day photo-treatment using five different markers. According to the literature, in all samples neoplastic cells stained positive for vimentin, S100 and PNL2, while negative for FVIII and pancytokeratin. In conclusion, visible light can be effectively exploited to bleach pigmented melanoma FFPE sections prior to perform routine histochemical and immunohistochemical stains.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Bleaching melanin in formalin-fixed and paraffin-embedded melanoma specimens using visible light: a pilot study

et al. 2019

View full text Add to dashboard Cite

show abstract

“…For this reason, based on the protocols recommended we decided to automate and standardize the use 0.5% H 2 O 2 Tris base pH 10 as bleaching buffer. This because it guaranteed an excellent compromise between bleaching efficacy, processing time and antigenic conservation compared with the other proposed buffers 10–14…”

Section: Discussionmentioning

confidence: 99%

A Fast and Automated Melanin-bleaching Method for Histopathologic Evaluation of Pigmented Melanoma Tissues

Ugolini

Baroni

Nassini

et al. 2021

Applied Immunohistochemistry &Amp; Molecular Morphology

View full text Add to dashboard Cite

Histopathologic examination of highly pigmented melanoma tissues has always been a challenge for pathologists. The high concentration of melanin pigment is an obstacle for immunohistochemistry and the ensuing evaluation. Therefore, removing melanin has become a crucial step for processing heavily pigmented melanoma samples. Several bleaching techniques have been proposed in the past, however, the most commonly used methods are time-consuming and poorly standardized. In this study, we propose a new fast and fully automated bleaching method applicable to validated immunohistochemical panels already used in the diagnosis of melanocytic tumors. The proposed bleaching protocol is based on sample pretreatment with 0.5% hydrogen peroxide and a Tris base pH 10 solution for 8 minutes at 80°C before antigen retrieval. Immunohistochemistry with HMB45, MART-1, Ki-67, SOX10, S-100, Tyrosinase, and BRAF(V600E) antibodies showed that this pretreatment removed excess melanin without affecting the tissue antigenicity and cytoarchitecture. In conclusion, we propose a new fast and automated bleaching protocol, easily transferable to a routine setting with efficient results in specimens in which the melanin pigmentation could blunt the histopathologic examination.

show abstract

“… Note: It might take 30–60 min for the hydrophobic barrier to dry out. Bleach the pigment of eyeball tissue by applying bleaching solution to sections and illuminate by direct cool light for 20 min (see our previously published method ( Jordan et al., 2019 )). Note: If the pigment bleaching is not required, apply 3% H2O2 for 15 min to inactivate the endogenous peroxidase enzyme activity (the direct light illumination is unnecessary).…”

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

“…Bleach the pigment of eyeball tissue by applying bleaching solution to sections and illuminate by direct cool light for 20 min (see our previously published method ( Jordan et al., 2019 )).…”

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

Step-by-step preparation of mouse eye sections for routine histology, immunofluorescence, and RNA in situ hybridization multiplexing

et al. 2021

Self Cite

View full text Add to dashboard Cite

Comparison of bleaching protocols utilizing hematoxylin and eosin stain and immunohistochemical proliferation marker MCM3 in pigmented melanomas

Cited by 11 publications

References 9 publications

Bleaching melanin in formalin-fixed and paraffin-embedded melanoma specimens using visible light: a pilot study

Bleaching melanin in formalin-fixed and paraffin-embedded melanoma specimens using visible light: a pilot study

A Fast and Automated Melanin-bleaching Method for Histopathologic Evaluation of Pigmented Melanoma Tissues

Step-by-step preparation of mouse eye sections for routine histology, immunofluorescence, and RNA in situ hybridization multiplexing

Contact Info

Product

Resources

About