Alkaline phosphatase red substrate

Immunohistochemistry was performed using a standard avidin–biotin complex–horse radish peroxidase (ABC‐HRP) method, and visualized with diaminobenzidine (Vector Laboratories, UK), except for albumin immunohistochemistry which was performed using a standard ABC–alkaline phosphatase method, and visualized with alkaline phosphatase red substrate (Vector Laboratories, UK). Both isotype controls and no primary antibody controls were included in every run. A summary of all the primary antibodies and their conditions of use is shown in Table 2. All immunohistological evaluation of both the human and mouse ageing cohorts was performed blind to any clinical information.

Paraffin-embedded tissue sections were processed for antigen retrieval by heat-induced epitope retrieval in citrate buffer pH 6 (DAKO, Glostrup, Denmark, cat. number S2367). After epitope retrieval, sections were then incubated with either rat anti-F4/80 (Invitrogen, cat. number MF48000) (1:1000 in blocking buffer TBS with 2% FBS) or rabbit anti-SLC48A1 (Zhang et al., 2018 (link)) (1:500) overnight at 4°C. Polyclonal SLC48A1 antibody serum was generated in rabbit using the C-terminal 17 amino acid peptide sequence (YAHRYRADFADIILSDF) of human SLC48A1 as antigen (Epitomics, Inc). Sections were then incubated with secondary biotinylated anti-rat antibody (Vector labs, cat. number BA-9400) for 30 min at room temperature. Signals were detected by DAB substrate or the alkaline phosphatase red substrate (Vector labs, cat. number: SK-5100) incubation and slides were lightly counterstained with hematoxylin. H and E and Perl’s Prussian blue stainings were conducted by Histoserve, Inc.

Paraffin-embedded tissue sections were processed for antigen retrieval by heat-induced epitope retrieval in citrate buffer pH 6 (DAKO, Glostrup, Denmark, cat. number S2367). After epitope retrieval, sections were then incubated with either rat anti-F4/80 (Invitrogen, cat. number MF48000) (1:1000 in blocking buffer TBS with 2% FBS) or rabbit anti-SLC48A1 (Zhang et al., 2018 (link)) (1:500) overnight at 4°C. Polyclonal SLC48A1 antibody serum was generated in rabbit using the C-terminal 17 amino acid peptide sequence (YAHRYRADFADIILSDF) of human SLC48A1 as antigen (Epitomics, Inc). Sections were then incubated with secondary biotinylated anti-rat antibody (Vector labs, cat. number BA-9400) for 30 min at room temperature. Signals were detected by DAB substrate or the alkaline phosphatase red substrate (Vector labs, cat. number: SK-5100) incubation and slides were lightly counterstained with hematoxylin. H and E and Perl’s Prussian blue stainings were conducted by Histoserve, Inc.