Journal: Infection and Immunity

Article Title: Mucosal Vaccination against Tuberculosis Using Inert Bioparticles

doi: 10.1128/IAI.00786-13

Figure Lengend Snippet: IFN-γ responses. IFN-γ was determined from splenocytes stimulated using an ELISA or ELISPOT assay. (a and b) ELISA detection of IFN-γ from splenocytes stimulated with rMPT64 (a) or with rAcr or rAg85B (b). (c) ELISPOT assay determination of the number of spot-forming units (SFU) in splenocytes stimulated with rMPT64 or rAcr-Ag85B. Spleens were taken from mice (n = 2) immunized using dosing regimen 1. The BCG group received one dose of BCG.

Article Snippet: Free binding sites were blocked with 10% FCS in PBS at RT for 1 h. Culture supernatants were tested at RT for 2 h, and IFN-γ was detected with a biotinylated anti-mouse IFN-γ–streptavidin–horseradish peroxidase conjugate (BD Biosciences) at RT for 1 h. Recombinant mouse IFN-γ (BD Biosciences) was used as a standard.

Techniques: Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal: Infection and Immunity

Article Title: Mucosal Vaccination against Tuberculosis Using Inert Bioparticles

doi: 10.1128/IAI.00786-13

Figure Lengend Snippet: Gating strategy for polyfunctional T cell analysis. Representative FlowJo scatter plots of a naive mouse splenocyte sample are presented to show the gating strategy. Samples were first gated by forward scatter (FSC-A)/side scatter (SSC-A) to select lymphocytes and were then gated by FSC-H/FSC-A to gate for single cells. This population was then gated for CD3+ CD4+ or CD3+ CD8+ staining. These two populations were then analyzed for IFN-γ, IL-2, and TNF-α by staining.

Article Snippet: Free binding sites were blocked with 10% FCS in PBS at RT for 1 h. Culture supernatants were tested at RT for 2 h, and IFN-γ was detected with a biotinylated anti-mouse IFN-γ–streptavidin–horseradish peroxidase conjugate (BD Biosciences) at RT for 1 h. Recombinant mouse IFN-γ (BD Biosciences) was used as a standard.

Techniques: Staining

Journal: Cell Stress & Chaperones

Article Title: An unfolded protein response is the initial cellular response to the expression of mutant matrilin-3 in a mouse model of multiple epiphyseal dysplasia

doi: 10.1007/s12192-010-0193-y

Figure Lengend Snippet: Histological and immunohistochemical analysis of wild-type and mutant xiphoid and rib cartilage. H&E staining of wild-type (wt) and mutant (m/m) a rib and b xiphoid cartilage growth plates from 5- and 21-day-old mice. Cells in the proliferative zone form ordered columns along the vertical axis of the GP ( black boxes ). In the m/m mice by 5 days of age, the chondrocytes ( red circles ) of the proliferative zone are rounder and more randomly orientated compared to the wt ( black box ). Scale bar is 100 µm. IHC with an anti-matrilin-3 antibody on c rib and d xiphoid growth plates from 5-, 14- and 21-day-old mice. In wild-type mice (wt), matrilin-3 staining was seen in the cartilage ECM at all ages. In mutant mice (m/m), matrilin-3 staining was predominantly within the chondrocytes with only minimal staining present in the ECM. Intracellular staining was observed from 5 days of age and was still present by 21 days of age

Article Snippet: Antibodies used were rabbit anti-matrilin-3 (R&D), rat anti-BrdU (Abcam) and goat anti-Grp94 (Santa Cruz).

Techniques: Immunohistochemical staining, Mutagenesis, Staining

Journal: Cell Stress & Chaperones

Article Title: An unfolded protein response is the initial cellular response to the expression of mutant matrilin-3 in a mouse model of multiple epiphyseal dysplasia

doi: 10.1007/s12192-010-0193-y

Figure Lengend Snippet: The effect of SPB treatment on the phenotype of mutant mice and the relative levels of matrilin-3 retention, chondrocyte proliferation and apoptosis. a Bone length measurements of treated and untreated mutant male mice were performed on radiographs taken at 9 weeks of age showed no significant differences. Measurements were made of the inner canthal distance ( ICD ), femur ( F ), pelvis ( P ) and tibia (T). ( n > 23 mice per group; nested ANOVA). b The body weights of male and female mutant mice showed no significant differences between untreated and treated groups. ( n > 23 mice; nested ANOVA). The relative levels of apoptosis in c the hypertrophic zone (HZ) and d at the VIF of the growth plate was calculated by counting the number of TUNEL-positive chondrocytes in the HZ or at the VIF compared to the total number of DAPI-stained chondrocytes in the growth plate. Although there appeared to be relatively less TUNEL-positive cells in the HZ and more apoptosis at the VIF in the treated group, these differences were not statistically significant. ( n > 20 sections per group; nested ANOVA). e Twenty-one-day-old mice were administered with 0.01 ml/g of the nucleotide analogue BrdU 2 h prior to sacrifice. IHC was performed on tibia sections using an anti-BrdU antibody. The relative levels of chondrocyte proliferation was determined by counting the number of BrdU-labelled nuclei compared to the total number of chondrocytes in the proliferative zone of the growth plate. There were no significant differences in the proportion of proliferating cells between the treated and untreated groups. ( n > 20 sections per group; nested ANOVA). f IHC using anti-matrilin-3 antibody on tibia growth plates from 21-day-old mutant mice either treated or untreated with SPB. In both groups, there is extensive intracellular retention of mutant matrilin-3 ( scale bar is 100 µm)

Article Snippet: Antibodies used were rabbit anti-matrilin-3 (R&D), rat anti-BrdU (Abcam) and goat anti-Grp94 (Santa Cruz).

Techniques: Mutagenesis, TUNEL Assay, Staining