Thick brain sections were cut at 70 µm from paraffin-embedded autopsy tissue using a sliding microtome (Jung, Heidelberg, Germany) as described previously [
27]. For topographical orientation, free-floating sections were pre-treated with performic acid and processed with aldehyde fuchsin for selective staining of lipofuscin pigment combined with a basophilic Nissl stain (Darrow red) [
7]. Antigen retrieval was performed by pre-treating the section with performic acid or in a steamer (95 °C) with 10 mM citrate buffer at pH 6.0 for 10–15 min. Endogenous peroxidase activity was inhibited with a mixture of 10% methanol and 3% concentrated H
2O
2 in Tris-buffered saline (TBS) for 30 min. Non-specific binding sites were blocked using 5% bovine serum albumin (BSA) for 30 min at room temperature. For the visualization of MCH neurons, sections were incubated with a primary antibody against pro-melanin-concentrating-hormone (PMCH) overnight at 4 °C (1:1000 polyclonal rabbit, HPA046055, Atlas Antibodies AB, Bromma, Sweden). The levels of PMCH in the LHA are expected to correspond to the expression of MCH, as PMCH prohormone is cleaved into MCH and neuropeptide glutamic acid-isoleucine (NEI) peptides, both expressed within the same neurons in human hypothalamus [
28]. The expression of neuropeptide-glycine-glutamic acid (NGE), which is a third mature peptide derived from the prohormone PMCH, has not been observed in the brain [
74]. ALS-related pathological protein aggregates were detected by incubating the sections with the primary anti-pTDP-43 antibody, which detects TDP-43 abnormally phosphorylated at S409/410 (1:4000 for IF, 1:10000 for IHC, polyclonal rabbit, TIP-PTD-P02, Cosmo Bio Co., Ltd, Tokyo, Japan). For single- and double-label IHC, sections were incubated with a secondary biotinylated antibody (1:200; 2 h, room temperature, Vector Laboratories, Burlingame, CA, USA) followed by incubation with the avidin–biotin-peroxidase complex (1½ hours, room temperature, ABC Vectastain kit, Vector Laboratories, Burlingame, CA, USA). For the visualization of the first primary antibody, 3,3´-diaminobenzidine tetrahydrochloride (DAB; Sigma Taufkirchen, Germany) was used as the chromogen. For double-label IHC, sections were further treated with TBS at 95 °C for 5 min, and the whole immunohistochemical procedure was repeated using the next primary and secondary antibody. Subsequently, a blue chromogen (Vector SK-4700 peroxidase substrate kit, Linaris, Doffenheim; Germany) was used to visualize the second reaction product. Finally, tissue sections were cleared, mounted, and cover-slipped in a medium with a refraction index of 1.58 (Histomount, Thermo Fischer Scientific, Braunschweig, Germany, plus 10% α-methyl-cinnamaldehyde). For immunofluorescence, sections were incubated with the corresponding secondary antibody (1:200, donkey anti-rabbit Alexa Fluor 594, ab150064, Abcam, Cambridge, UK, 1:200) for 1 h at room temperature. Cell nuclei were visualized with 4′, 6-diamidino-2-phenylindole (DAPI) and Mowiol was used as a mounting medium. Additional 7 µm-thick paraffin sections were cut from paraffin blocks and mounted on object slides to investigate the presence of hypothalamic di-peptide repeat (DPR) pathology. For this purpose, sections were deparaffinized and treated with 3% H
2O
2 in TBS for 20 min. For antigen retrieval, citrate buffer was applied at 100 °C for 20 min. Sections were incubated overnight with the primary mouse antibody against the poly-GA peptide repeat sequence found in C9ORF72/C9RANT (1:2000, Merck Chemicals GmbH, Darmstadt, Germany), followed by incubations with a biotinylated anti-mouse secondary antibody (1:200 for 2 h, Vector Laboratories, Burlingame, CA, USA), and the ABC Vectastain kit (1½ hours, room temperature, Vector Laboratories). The reaction product was visualized with DAB or SK-4700, and the sections were counterstained with Haemalaun or Darrow red, respectively. In negative controls, the absence of the primary antibody incubation resulted in the absence of staining for all markers.