Depex

GUS activity in transformed roots and nodules was analyzed using 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid as a substrate. Roots and nodules were vacuum infiltrated during 20 min and subsequently incubated in a GUS buffer at 37 °C. Incubation lasted 6 h and 10 h for pMtNRLK1-GUS and pMtSUNN-GUS, respectively. After staining, roots and root nodules were fixed, dehydrated, embedded with Technovit 7100 (Heraeus Kulzer, Wehrheim, Germany), according to the manufacturer’s instructions, and sectioned with a microtome (Reichert-Jung, Nussloch, Germany). Three μm thick sections were mounted on coated slides (Sigma-Aldrich, St. Louis, MO). For tissue counter staining, sections were treated with a 0.05% (w/v) ruthenium red solution (Sigma-Aldrich, St. Louis, MO), washed in distilled water, and dried. Sections were mounted with Depex (BDH Chemicals, Poole, England), and pictures were taken with a Diaplan microscope equipped with bright- and dark-field optics (Leitz, Wetzlar, Germany).

Four micrometer-thick paraffin sections of the middle temporal gyrus were used for immunohistochemistry. After rehydration and antigen retrieval, including neat formic acid pre-treatment and heat-induced epitope retrieval, sections from AD and iAD cases were stained with rabbit-anti-human Aβ43 (IBL, Fujioka, Japan; cat. no. 18583, 0.5 μg/ml). Furthermore, sections were stained with mouse-anti-human Aβ42 (clone 21F21, 1:4000) and mouse-anti-human Aβ40 (clone 2G3, 1:4000), both provided by Elan Pharmaceuticals (South San Francisco, CA, USA) [16 (link)]. Binding of biotinylated secondary antibody (goat-anti-rabbit or rabbit-anti-mouse, DAKO, Glostrup, Denmark) was detected with the Vectastain ELITE ABC kit (Vector Laboratories, Peterborough, UK), using 3,3′ diaminobenzidine (DAB) as chromogen and 0.05% hydrogen peroxide as substrate. Sections were mounted in DePex (BDH Laboratory Supplies, Poole, UK).

Immunohistochemistry (IHC) was used to detect the presence of Ki67. Briefly, sections were dewaxed with xylene and rehydrated using decreasing concentrations of ethanol and ddH₂O. Slides were washed in Tris Buffered Saline (TBS: 0.1 M Tris pH 7.5 and 0.3 M NaCl) with 0.05% Tween-20 (TBST). Endogenous peroxidase was quenched by washing with 3% H₂O₂ (Fisher Scientific, Loughborough, United Kingdom) in Phosphate Buffered Saline (PBS) for 5 min. Sections were blocked with 1.5% normal goat serum (Vectastain ABC Kit, Vector Labs, Peterborough, United Kingdom) in TBS for 1 h and then incubated with rabbit anti-Ki-67 antibody (ab66155; Abcam, Cambridge, United Kingdom) at 1:100 overnight at 4°C; control sections were incubated with normal goat serum 1.5%. After 3 washes with TBST, sections were incubated with biotinylated anti-rabbit IgG secondary antibody (Vectastain ABC Elite Kit, Vector Laboratories) for 1 h, followed by ABC solution (Vectastain ABC Elite Kit, Vector Laboratories) for 30 min. A 3,3′-Diaminobenzidine (DAB) peroxidase substrate kit (Vectastain ABC Elite Kit, Vector Laboratories) was used to develop the stain. The slides were counterstained with haematoxylin, dehydrated, mounted with DEPEX (VWR, Leicestershire, United Kingdom) and imaged. For analysis of Ki67 staining, GCs were classified as either Ki67 negative or positive using an assessment scale (see results).

Thawed sections were fixed with paraformaldehyde (4%, 20 min), washed in PBS (2 × 5 min), incubated in pre‐chilled 3% H₂O₂ in methanol (20 min), and then washed in PBS (2 × 5 min). Each section was blocked with normal goat serum (NGS; 10%; 30 min; #S‐1000; Vector Laboratories, CA, USA), incubated with AVIDIN blocking solution (15 min; #SP‐2001; Vector Laboratories), rinsed in PBS, and then incubated with rat anti‐mouse CD68 primary antibody (1:200; 5% NGS; 4°C; #mca1957ga; Bio‐Rad, NSW, Australia) overnight. The slides were then washed in PBS (2 × 5 min) before being incubated with the secondary antibody (1:100; 5% NGS; 30 min; #BA‐4000; Vector Laboratories). Next, the sections were washed in PBS (2 x 5 min) and incubated with ABC avidin/biotin complex (30 min; PK‐6100; Vector Laboratories) and DAB solution (#SK‐4100; Vector Laboratories). Staining was terminated with distilled water. The sections were counterstained with Mayer Haematoxylin for 15 s and rinsed in tap water before blueing in Scotts tap water and washing in tap water. Finally, slides were dehydrated in ethanol (95% 3 min, 100% 3 × 3 min), cleared in xylene (2 × 5 min) and mounted with depex (#13515; VWR International, USA). Sections were imaged on the Olympus FSX100 microscope 4.2 × magnification, and images were analysed using Adobe Photoshop CC.

Tissue was fixed in 4% PFA and embedded in paraffin wax. Five-micrometre sections were stained with haematoxylin and eosin (H&E) using the Leica Autostainer and mounted in DePeX (VWR, Lutterworth, UK). Adipocyte diameter and pancreatic β-cell islet number and size were quantified using ImageJ software as previously described [7 (link), 73 (link)].

Six micrometre sections of formalin-fixed paraffin-embedded tissue from the TMA blocks were immunostained. The appropriate antigen retrieval steps were performed prior to addition of the primary antibodies. The list of primary antibodies used in the study is presented in Supplementary Table 2. Visualization of biotinylated secondary antibodies (Dako, Denmark) was achieved by using the avidin–biotin–peroxidase complex method (Vectastain Elite) with 3,3′-diaminobenzidine as the chromogen (Vector Laboratories, UK). The TMA sections were then counterstained with haematoxylin, dehydrated and mounted on DePeX (VWR International, UK). Appropriate negative controls were performed to ensure the specificity of the staining.