Sudan black b

To retrieve CRMP5 antigen and to reduce the masking effect of formalin fixation, the tumor tissues were treated as follows. Sections were immersed for 45′ in a Citrate Buffer antigen retrieval Solution (CBS, composed by 10 mM citric acid, 0.05% Tween 20, pH 6.0), preheated in a water bath at 95–98 °C, and then removed from the bath allowing slides to cool for 30 min at RT. Sections were washed three times in phosPB (10′ each) and incubated with primary antibodies, as described. To remove human-tissue auto-fluorescence caused by the presence of lipofuscin granules in the central nervous system, at the end of the immunofluorescence procedures, sections were treated with Sudan Black B. Following incubation in DAPI (Sigma-Aldrich, Cat. #D9542, concentration 1:4000) for 10 min, sections were rinsed three times in a phosphate buffer (PB), and then immersed in 0.3% Sudan Black B (Sigma Aldrich) in ethanol 70% for 25–30 s at room temperature, followed by another 3 consecutive washing steps in PB (10′ each).

Livers from subset of wild type (n = 4) and heterozygous (n = 4) mice with macro and/or microvesicular steatosis scores of 0–3 were stained with Oil Red O or Sudan Black B to confirm presence of steatosis. Briefly, cryosectioned livers were washed with running tap water for 10 minutes, rinsed with 60% isopropanol and stained with Oil Red O (Sigma, Cat# O0625-25G) mixed with 60% isopropanol for 15 minutes. The sections were then rinsed with 60% isopropanol, and nuclei were stained with Mayer’s Haematoxylin, rinsed in tap water and coversliped with aqueous mounting medium. For Sudan Black B staining, cryosectioned livers were washed in tap water for 10 minutes, rinsed in 70% ethanol for 1 minute and stained with Sudan Black B (Sigma, Cat# 199664-25G) diluted in 70% ethanol for 8 minutes. The sections were dipped in 70% ethanol for 2 minutes to remove any extra stain, and the nuclei were stained with 0.1% nuclear fast red. Sections were then washed in tap water for 10 minutes and coversliped with aqueous mounting medium. All samples were imaged using Leica microsystem (model DM6000B) and Leica camera (model DFC 450 C).

The producing PHB bacterial strain was isolated from a soil sample collected from the botanic garden of Skikda University by performing serial dilutions. Pure, viable colonies were then grown in a mineral salt medium (MSM) [20] (link) to which 2% glucose and 10 g of agar were added; the mixture was then incubated for 48 h at 37 °C. The PHB-producing colonies were detected by adding an ethanolic solution of 3% Sudan Black B (C29H24N6 224-087, Sigma). The Sudan Black B positive isolate was tested for its ability to produce PHB in the agar MSM medium [20] (link) and added 2% of carob molasses instead of glucose. The bacterial strain was spotted onto the MSM agar medium and incubated at 37 °C for 48 h. The bacterial colonies were colored with an ethanolic solution of 3% Sudan Black B (C29H24N6 224-087, Sigma) to confirm its ability to produce PHB [21] (link).

Lipofuscin was detected by Sudan black B staining (380B, Sigma‒Aldrich, Merck) following the manufacturer’s instructions. Briefly, the cells were fixed with 4% paraformaldehyde, stained with Sudan black B solution, and finally labeled with Nuclear Fast red (1.15939, Sigma‒Aldrich, Merck). The cells were observed with a phase-contrast microscope.

The selected slides were first dehydrated in a series of graded alcohols. They were then placed in a plastic Coplin jar with 10 mM citrate buffer, pH 6, inside a pressure cooker for 20 min to recover the antigen. After this process, tissues were permeabilized with PBST (0.01 M PBS and 0.1% Triton) for 30 min and nonspecific sites were blocked with 5% horse serum in PBST in a humid chamber for 30 min. The tissues were then incubated with rabbit anti-NeuN primary antibody (1:500, No. Cat. D4G40, Cell Signaling, Danvers, MA, USA) in a humid chamber at 4 °C overnight. Subsequently, tissues were washed with PBST and incubated with Alexa Fluor^® 488 donkey anti-rabbit secondary IgG antibody (1:500, Molecular Probes, Eugene, OH, USA) for 2 h at room temperature in the dark. The slides were then placed in a 0.1% black Sudan B (Sigma-Aldrich, St. Louis, MI, USA) solution in 70% ethanol for 15 min, after which the excess black Sudan B was discarded with PBS. The tissues were covered with Vectashield (Vector Laboratories, CA, USA) and a coverslip. The slides were observed with the Aperio FL fluorescence scanner (Leica, Biosystems, Pleasanton, CA, USA) at 1× and magnifications were taken at 20×. Specific fluorescence was quantified in the spinal cord tissue’s rostral, epicentral and caudal areas. Cells colocalized with NeuN and DAPI were quantified manually.

Formalin-fixed and paraffin-embedded brain sections of frontal cortex were dewaxed and pretreated with Tris/EDTA buffer pH 9 at high temperature. The following primary antibodies were incubated overnight at 4 °C: polyclonal goat anti-YKL-40 (R&D Systems, AF2599, dilution 1:200), rabbit anti-GFAP (Sigma, G9269, dilution 1:500), phosphorylated tau clone AT8 (Thermo Scientific, MN1020, dilution 1:1000), monoclonal mouse anti-MAP2 (Sigma, M4403, dilution 1:500) and rabbit anti-Iba-1 (Wako Chemicals, 019-19741, 1:500). For IHC, the endogenous peroxidase activity was blocked, sections were HRP-labelled (Dako, Glostrup, Denmark, dilution 1:200) and the reaction was visualized by the EnVision+ system peroxidase procedure (DAKO, Glostrup, Denmark). For IF, sections were incubated for 1 h with Alexa Fluor 488, 555 or 647 (Invitrogen, Carlsbad, CA, USA, dilution 1:1000) secondary antibodies and stained with Sudan black B (Merck, Whitehouse Station, NJ, USA) to mask tissue autofluorescence. Nuclei were stained with Hoechst 33258 (Life Technologies, Carlsbad, CA, USA, dilution 1:1000), and coverslips were added with Immu-Mount (Fisher Scientific, Rockford, USA) mounting medium.