Immunohistochemical Analysis of Buruli Ulcer Biopsies

Fixed and embedded 4 mm punch biopsies from Buruli ulcer patients were described previously [6 (link)]. In ulcerated BU lesions (6 patients) punch biopsies were taken 1 cm inside the outer margin of the induration surrounding the ulcer. For BU plaque lesions (2 patients) punch biopsies were collected from the non-ulcerated center of the lesion. Healthy control tissue was obtained at The Whitely Clinic or purchased from AMS Biotechnology (Europe) Ltd (500041028). After removal, all tissues were fixed in neutral formalin, embedded into paraffin (pfm Medical, 9000R2010) and sectioned (5 μm). Sections were deparaffinized, endogenous peroxidase quenched, epitopes unmasked (pH 6 citrate buffer; Sigma, C999) and blocked with horse serum (Vector Laboratories, S-2000-20). The tissue sections were then incubated with either anti-SQSTM1 antibody (Abcam, ab56416) or IgG2a isotype control (Santa Cruz Biotechnology, sc-3878) overnight at 4°C and biotin-conjugated secondary antibody (Vector Laboratories, BA2000). Staining was performed using VECTASTAIN Elite ABC kit (Vector Laboratories, PK-6100) and Vector NovaRED peroxidase substrate (Vector Laboratories, SK-4800). Counterstaining was performed with Shandon Harris Hematoxylin (Thermo Fisher Scientific, 6765001).

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Hall B.S., Dos Santos S.J., Hsieh L.T., Manifava M., Ruf M.T., Pluschke G., Ktistakis N, & Simmonds R.E. (2021). Inhibition of the SEC61 translocon by mycolactone induces a protective autophagic response controlled by EIF2S1-dependent translation that does not require ULK1 activity. Autophagy, 18(4), 841-859.

Publication 2021

pH 6 citrate buffer horse serum ab56416 IgG2a isotype control biotin-conjugated secondary antibody VECTASTAIN Elite ABC kit Vector NovaRED peroxidase substrate Shandon Harris Hematoxylin

Anti antibody Antibody Biopsies Biotin Buffer Buruli ulcer Citrate Embedded paraffin Epitopes Formalin Hematoxylin Horse Igg2a Isotype Patients Peroxidase Plaque Serum Tissue Vector

Corresponding Organization : University of Surrey

Other organizations : Babraham Institute, Swiss Tropical and Public Health Institute, University of Basel

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Variable analysis

independent variables

Location of punch biopsy collection (1 cm inside the outer margin of the induration surrounding the ulcer for ulcerated BU lesions, or from the non-ulcerated center of the lesion for BU plaque lesions)

dependent variables

SQSTM1 (p62) protein expression

control variables

Healthy control tissue obtained from The Whitely Clinic or purchased from AMS Biotechnology (Europe) Ltd
Formalin fixation and paraffin embedding of all tissue samples
Sectioning of tissue samples at 5 μm thickness
Deparaffinization, endogenous peroxidase quenching, epitope unmasking, and blocking with horse serum prior to immunohistochemical staining
Overnight incubation of tissue sections with anti-SQSTM1 (p62) antibody or IgG2a isotype control at 4°C
Incubation with biotin-conjugated secondary antibody
Staining using VECTASTAIN Elite ABC kit and Vector NovaRED peroxidase substrate
Counterstaining with Shandon Harris Hematoxylin

positive control

IgG2a isotype control

negative control

Healthy control tissue

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