Ab104704

The following primary antibodies were used: mouse monoclonal anti-CD26 (mAb M-A261, Invitrogen) and rabbit polyclonal (pAb) anti-CD26 antibody (Abcam, Ab28340). Other antibodies used in the present study were rabbit pAb anti-apolipoprotein-E (ApoE; Abcam, Ab85311), anti-CD3 (PA5-32318) Thermo Fisher Scientific, anti-cluster of differentiation 42b (CD42b; Abcam, Ab104704), anti-rabbit polyclonal glycophorin (Abcam, 196568), anti-divalent metal protein 1 (DMT1; Abcam, Ab123085), anti-ferroportin (FPN; Abcam Ab85370), anti-GFAP (Abcam Ab48050), anti-CD68 (Sigma–Aldrich, AMAB98073), and anti-CD11b (Thermo Fisher, mAbM1/70). The following secondary antibodies were used: biotinylated goat anti-rabbit-Ig and biotinylated horse anti-mouse (both from Vector Laboratories, 1:250 for IHC); Alexa Fluor 568-labelled donkey anti-mouse-Ig, Alexa Fluor 488-labelled donkey anti-rabbit-Ig, and Alexa Fluor 568-labelled donkey anti-goat-Ig (all from Invitrogen, 1:1000 for immunofluoresence).

A polyclonal rabbit anti-hepcidin 25 (Abcam, ab30760), recognising a 2.8 kDa protein [53 (link)], monoclonal antibody (MAB) human anti-ferritin light chain (Abcam, ab69090), human anti-ferritin heavy chain (Abcam, ab65080), anti-DMT1 (Abcam, ab123085), anti-FPN (Abcam, ab85370), anti-CD42b (Abcam, ab104704), anti-rabbit polyclonal glycophorin (Abcam, ab196568), anti-GFAP (Abcam, ab48050), anti-CD68 (Sigma-Aldrich, AMAB98073), MAB anti-CD11b (Thermo Fisher, Waltham, MA, USA, mAbM1/70), GFAP (Abcam, ab48050), MAB anti-Iba1 (Thermo Fisher, MAB M1/70), polyclonal anti-Iba1 (Wako, Fujifilm, Tokyo, Japan, catalogue number 019-19741), MAB anti-IL-6 (Thermo Fisher, catalog number M620), MAB IL-1β (Thermo Fisher, ILB1-H67), MAB anti-β amyloid 42 (Covance, Princeton, NJ, USA, SIG 39320), anti-phospho-tau (AT8, Thermo Fisher, MN1020), SOD1 (Abcam, ab252426), S100β (Abcam, ab218514), RUNX1 (Sigma-Aldrich, HPA004176) and OLIG2 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-365644) were used for IHC or Western blotting. The following secondary antibodies were used: biotinylated goat anti-rabbit-Ig and biotinylated horse anti-mouse (both from Vector Laboratories, 1:250 for IHC), Alexa Fluor 568-labelled donkey anti-mouse-Ig, Alexa Fluor 488-labelled donkey anti-rabbit-Ig and Alexa Fluor 568-labelled donkey anti-goat-Ig (all from Invitrogen, 1:1000 for immunofluoresence).

Three sections (100 µm intervals) were prepared from the middle portion of the thrombus and immunohistochemically stained. For the immunological staining of fibrin(ogen), anti-fibrin(ogen) antibody (1:200, ab63983; Abcam, Cambridge, MA, USA) and Alexa Fluor 488–conjugated secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) were used. For Ly-6G, Alexa Fluor 647–conjugated anti-mouse Ly-6G (1:400, BD127626; BioLegend, San Diego, CA, USA) was used. For CD42b, anti-CD42b antibody (1:1000, ab104704; Abcam) and Alexa Fluor 488–conjugated secondary antibody (Thermo Fisher Scientific) were used. For Cit-H3, anti-Cit-H3 antibody (1:250, ab5103; Abcam, Cambridge, MA, USA) and Alexa Fluor 488–conjugated secondary antibody (Thermo Fisher Scientific) were used. Nuclei were labeled using 4’, 6-diamidino-2-phenylindole (excitation wavelength: 360 nm, fluorescence wavelength 461 nm, 62248; Thermo Fisher Scientific) or SYTOX Green (excitation wavelength: 504 nm, fluorescence wavelength 523 nm, 33025; Thermo Fisher Scientific). Sections were examined using an LSM 510 META confocal microscope (Carl Zeiss, Jena, Germany). Non-immune rabbit immunoglobulin G and polyclonal isotype control were used as negative controls of fibrin(ogen), CD42b, and Cit-H3. Positive staining was evaluated using ImageJ software v1.50i (https://imagej.nih.gov/ij/index.html, accessed on 20 October 2021).