Protein block serum free ready to use

CDCs cultured on 24-well culture dishes were fixed and blocked with Protein Block Serum-Free Ready-to-Use (Dako) for 1 h. Then they were incubated with rabbit monoclonal antibody against gH2AX antibody (Ser 139, #9718, Cell Signaling Technologies) for 1 h at room temperature. Then they were washed and incubated with a DyLight 550-conjugated goat anti rabbit IgG antibody (ab96884, Abcam). Next, they were incubated with rabbit monoclonal antibody against Ki67 antibody conjugated with Alexa Fluor 488 (ab197234, Abcam) for 1 h at room temperature. Nuclei were stained with DAPI. Positively stained cells were counted by using BZ-X710 All-in-One fluorescence microscope (KEYENCE). CDCs in each sample were classified by the number of gH2AX foci per nucleus (gH2AX foci per nucleus = 1, 2, 3 or ≥4) and the number of cells belonging to each category was counted. Also, the number of gH2AX positive cells in Ki67-negative cells was counted. In this particular experiment, only samples #1 to #25 were used, and #26 was not scored.

Mice were given flush-perfusion with phosphate-buffered saline (PBS) followed by perfusion-fixation with 4% paraformaldehyde (PFA). The removed brain was immersed in 4% paraformaldehyde and subsequently in 30% sucrose for 48 h at 4 °C. After the brain had been cut into coronal 30-μm sections, H₂O₂-inactivation of endogenous peroxidase activity was performed for immunohistochemistry, followed by treatment with protein block serum-free ready-to-use (Dako, Santa Clara, CA, USA) for 1 h at room temperature to block non-specific protein binding. The sections were incubated with a mouse monoclonal antibody against FcεRIα (9E1, ab54411, abcam^®, Cambridge, UK) in antibody diluent with background reducing components (Dako, Santa Clara, CA, USA) for overnight at 4 °C. After incubation with an HRP-conjugated secondary antibody, the sections were then treated with 3,3′-diaminobenzidine (DAB) for immunohistochemistry.

The immunohistochemical expression of LAG-3 was analyzed using a tissue microarray (KIN-2, AZUMAYA, Tokyo, Japan). Tissue microarrays consisted of paraffin-embedded tissue blocks constructed by extracting 3-mm cylindrical tissue cores, with appropriate histological findings. All blocks were sliced into Sect. (4 μm thick). After deparaffinization and heat-induced antigen retrieval using 1 mM ethylenediaminetetraacetic acid (pH 9.0), all sections were incubated with 3% hydrogen peroxidase for 10 min to quench the endogenous peroxidase activity. These sections were further incubated with DAKO Blocking Reagent (Protein Block Serum-Free Ready-to-use [Code X0909], DAKO North America Inc., California, USA) at room temperature for 20 min to block nonspecific antigens. LAG-3 antibody (Ab180187, clone EPR4392, diluted 1:1,500; Abcam) was applied as the primary antibody at 4 °C overnight. Secondary antibodies (HISTOFINE Simple Stain MAX-PO MULTI [NICHIREI Code 424,151], Nichirei Biosciences Inc., Tokyo, Japan) were applied at room temperature for 45 min. The antigen/antibody complex formation was performed in 3,3ʹ-diaminobenzidine tetrahydrochloride and hydrogen peroxidase substrate solution for 10 min and counterstained with hematoxylin for 5 s.

For immunocytochemistry, culture cells were fixed with 4% paraformaldehyde (PFA) in PBS for 10 min at 4 °C. After washing with PBS, they were treated with 0.1% Triton X-100 (Sigma-Aldrich, #T8787-100 ML) or PBS for 10 min at 4 °C, depending on the targeted antigen localization. After washing with PBS, cells or tissue sections were incubated with Protein Block Serum-Free Ready-To-Use (Dako, #X 0909) for 30 min at room temperature, followed by reaction with primary antibody in blocking buffer over night at 4 °C. After washing with PBS, the sections were incubated with fluorescent-conjugated secondary antibody. Anti-rabbit or anti-goat immunoglobulin G (IgG) bound to Alexa 488 or 546 was incubated in blocking buffer for 30 min at room temperature. The nuclei were stained with -Cellstain®- DAPI solution (1:1000, DOJINDO, Japan, D523). For immunohistochemistry, the tissue sections embedded into paraffin were deparaffinized and washed with PBS. Slides were incubated with primary antibodies, followed by further incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (ZSJQ-Bio, Beijing, China, 1:100). The antibody stains were developed by the addition of diaminobenzidine (DAB), and the nucleus was stained with hematoxylin. All images were captured using fluorescence microscopy (BZ-X700, KEYENCE). Antibody information is provided in Additional file 2: Table S1.

Cells were fixed with 4% paraformaldehyde for 10 min at room temperature. After being washed with PBS and treated with 0.1% Triton X-100 (Nacalai tesque) for 10 min, they were exposed to Protein Block Serum-Free Ready-To-Use (DAKO, Denmark) for 30 min at room temperature, and then incubated overnight at 4 °C in primary antibodies diluted with 1% BSA. Following washing with PBS, they were incubated for 30 min at 4 °C in secondary antibodies (1:500 diluted with BSA) and DAPI (1:1000 with BSA). Then, they were mounted with Fluorescence Mounting Medium (DAKO). Antibody information is provided in Table 2.Table 2

Antibody list

Primary antibody
MITF	M362129	(DAKO)
Tyrosinase	ab738	(Abcam)
MelanA	M719629	(DAKO)
CK14	ab7800	(Abcam)
TRP1	ab235447	(Abcam)
DAPI	D523	(Dojindo)
Secondary Antibody
Goat anti-mouse IgG1 AF546	A21123	(Invitrogen)
Goat anti-mouse IgG3 AF594	A21155	(Invitrogen)
Goat anti-mouse IgG2a AF594	A21135	(Invitrogen)
Goat anti-rabbit IgG(H + L) AF488	A11008	(Invitrogen)
Goat anti-Mouse IgG (H + L) AF546	A11003	(Invitrogen)