Cd68 pgm 1

Formalin-fixed, paraffin-embedded brain and spinal cord tissue was available from selected regions (table 1). The paraffin sections were cut at 5 μm, mounted on glass slides, and stained with routine hematoxylin and eosin. Representative 5-μm sections were immunostained for TDP43, β-amyloid, α-synuclein, hyperphosphorylated tau, p62, and CD68 with the following antibodies: TDP43 (2E2-D3, 1:3,000, Abcam, Cambridge, UK), β-amyloid (6F3D, 1:50, DAKO, Glostrup, Denmark), α-synuclein (KM51, 1:50, Leica/Novocastra, Buffalo Grove, IL), AT8, (MN1020, 1:100, Invitrogen, Carlsbad, CA), p62 (3/P62LCK Ligand, 1:100, BD Transduction, East Rutherford, NJ), and CD68 (PG-M1, 1:100, DAKO), respectively. Immunostaining was performed on either a BondMax autostainer (Leica Microsystems, Wetzlar, Germany) or a Roche (Basel, Switzerland) Ventana Discovery automated staining platform following the manufacturer's guidelines, using biotinylated secondary antibodies and a horseradish peroxidase–conjugated streptavidin complex and diaminobenzidine as a chromogen. All immunostainings were carried with appropriate controls. Gliosis, microglial activity, and the density of TDP43 pathologic inclusions were scored semiquantitatively.

A subset of cases was analyzed histopathologically. Thymic tissue of five SIDS and five control cases (age-matched) was formalin-fixed and paraffin-embedded. Analysis of the distribution of immune cells was performed by light microscopy. Therefore, hematoxylin-eosin (HE) staining and IHC of all 10 cases was done, including CD4 and CD8 (both Roche, Basel, Switzerland) for T lymphocytes, CD 138 (Dako, Glostrup, Denmark) for plasma cells and CD68-PGM-1 (Dako, Glostrup, Denmark) for macrophages.

Formalin-fixed, paraffin-embedded (FFPE) tumor tissues obtained from each patient before the vaccination were analyzed. Serial 2-μm thick sections were cut and processed for IHC. Primary antibodies used: CD3-Policlonal (A045201–2, 1:400), CD8 (C8/144B, 1:20), CD68-KP1 (M081401–2, 1:3000) and CD68-PGM-1 (M087601–2, 1:50), all purchased from Dako; CD163-10D6 (NCL-CD163, 1:200), GZMB (11F1, 1:80) (Novocastra, Leica Biosystems); Tbet (4B10, 1:80) (Santa Cruz); MHC-I-EMR8–5 (Ab70328, 1:4000) (Abcam); NY-ESO-1-E978 (35–6200, 1:200) (Thermofisher); PD1-NAT105 (3137, 1:50) (Biocare) and PDL1-RBT (BSB2654, 1:50) (Bio SB). All these primary antibodies were processed using the Autostainer Link 48 Dako System.
Immune infiltrating cells were quantified by counting the number of immune reactive cells at 400X magnification. The three areas with the most intratumor inflammatory cells were selected. Results are reported as mean value of immune reactive cells/mm².