Prolong gold mounting media

Transfected HEK293 cells were fixed with cold methanol:acetone (80%:20%) for 20 minutes. Cryosections were prepared from whole eyes fixed for 2h with 4% paraformaldehyde at 4°C collected at post-natal day 30 and underwent IF as described previously [10 (link)]. Briefly, after blocking in blocking buffer (1x PBS, pH 7.4, 1% fish gelatin, 2% donkey serum, 0.5% Triton X-100, 50 mg/ml bovine serum albumin), sections or cells were incubated overnight in primary antibodies in blocking buffer, followed by washing (4x 15 min with 1x PBS, pH 7.4), incubation in appropriate fluorescently labeled secondary antibodies in blocking buffer, additional washing, and mounting using ProlongGold mounting media (Life Technologies). Sections with a secondary antibody treatment only were used as a control to ensure the specificity of our antibodies and to normalize for background autofluorescence. All sections were treated with a 1% sodium borohydride solution for 2 min to minimize autofluorescence common in the retina. Cells and sections were imaged using an Olympus BX62 upright microscope equipped with a spinning disc confocal unit. Image analysis was performed using Slidebook v5 software (Intelligent Imaging Innovations, Denver, CO).

Cultures were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature and permeabilized with 0.1% Triton X-100 in PBS. Cells were blocked with 10% donkey serum, 0.1% Triton X-100 in PBS at room temperature and then incubated in primary antibody (2% donkey serum, 0.1% Triton X-100 in PBS) at 4º C overnight. The following antibodies were used: mouse anti-MAP2 at 1:500 (Chemicon), rabbit anti-GFAP (1:400 Chemicon), and mouse anti-NeuN at 1:400 (Milipore). Next day samples were incubated with secondary antibodies (anti-rabbit Alexa Fluor 488 and anti-mouse Alexa Fluor 568, both from Molecular Probes at 1:400 dilution) for 1 h, stained cell nuclei with Hoechst (Molecular Probes, 1:1000) washed and mounted in Prolong Gold mounting media (Life Technologies). Fluorescent wide-field images were acquired with Zeiss AxioImager.M1 microscope equipped with AxioCam HR camera and 40× objective ECPlan-Nerofluar/0.75/Ph2. For image analysis, we used custom-made plugin for ImageJ software. For analysis presented in Fig. 2b we quantified the number of nuclei positive for both NeuN and Hoechst immunoreactivity. For data shown in Fig. 2f we measured average intensity of KCC2 immunoreactivity in NeuN-positive cell bodies.

Spleen tissue sections with germinal centers (Fig. 3 and S4 Fig.) were incubated for 2h at 20°C with rat anti-mouse Foxp3 (1:100, Clone FJK-16s, Affymetrix) and hamster anti-MFGE8 (1:100, clone 18A2-G10, MBL) followed by 45 min with F(ab)2 AF594-conjugated donkey anti-rat IgG (1:400, Jackson ImmunoResearch) and Cy3-conjugated goat anti-Armenian hamster IgG (1:400, Jackson ImmunoResearch). The sections were then blocked 15 min with nonspecific rat IgG2a (1:30, Clone R35-95, BD Biosciences) and incubated 60 min with AF488-conjugated rat anti-human/mouse B220 (1:100, clone RA3-6B2, Affymetrix), biotinylated rat anti-mouse CD4 (1:100, clone RM4-5, Affymetrix) and PerCP-eFluor710-conjugated rat anti-mouse IgD (1:100, clone 11–26c, Biolegend). The sections were then incubated 30 min with Atto425-conjugated streptavidin (1:300, ATTO-TEC). Nuclei were stained with DAPI (2 μg/ml for 4 min) and the slides were mounted with ProlongGold mounting media (Life Technologies).

Kidneys were fixed post-mortem by immersion in 4% PFA overnight and then transferred to PBS with 30% sucrose and 0.05% sodium azide for 24 h. Tissues were then frozen and 40 μm sections cut using a Leica CM1900 cryostat. After three 5 min washes in PBS, sections were blocked at room temperature for 1 h in blocking buffer (0.3% Triton, 1% BSA, 1% donkey serum in PBS). The same buffer was used for the primary antibody incubation overnight at 4°C with gentle shaking. Sections were then washed 3 times in PBS for 10 min and incubated with Alexa Fluor secondary antibodies (donkey anti-rabbit and donkey anti-goat, 1:500) for 2 h at room temperature. After three 10 min washes in PBS, sections were mounted on glass slides using Prolong Gold mounting media (Life Technologies).

Immunofluorescence staining was performed in 3μm thick tissue sections adhered to silanized slides. After heat-induced antigen retrieval, samples were permeabilized and blocked in a single step incubating them for 30 minutes in PBS with 2% normal pig serum and 0.5% Triton X-100 (Research Organic, Cleveland, OH, USA) at room temperature (RT). Afterwards samples were washed and incubated at 4°C overnight with the primary antibodies: anti-CC16 (anti-Clara Cell Protein Secretory Antibody, Millipore cat 07–623 Merck KGaA, Darmstadt, Germany) and anti-prosurfactant Protein C (ab40879, Abcam, Cambridge, UK). After incubation, samples were washed and incubated with fluorescent secondary antibodies (Alexa Fluor 546 conjugated Donkey Anti-Mouse IgG and Alexa Fluor 647 conjugated Donkey Anti-Rabbit IgG; Jackson Immunoresearch, West Grove, PA) for 1 hour at RT. Samples were mounted with ProLong Gold mounting media (Life Technologies). All experiments included controls with/without primary antibody or with/without secondary antibody. Image capture was done with an FV-1000 confocal laser scanning microscope (Olympus Corporation, Tokyo, Japan) in sequential scanning mode to image each fluorochrome individually.