Journal: PLoS ONE

Article Title: Staphylococcus aureus Activates the NLRP3 Inflammasome in Human and Rat Conjunctival Goblet Cells

doi: 10.1371/journal.pone.0074010

Figure Lengend Snippet: Human conjunctiva was analyzed by immunohistochemistry and NLRP3 was shown to be highly expressed in the epithelium as indicated by pink staining (A). Arrows indicate epithelial layer of conjunctiva; arrowheads indicate goblet cells. Isotype controls were negative. Rat conjunctiva was analyzed by immunofluorescence microscopy (B) and all three of the inflammasome components NLRP3, caspase 1, and ASC were identified as demonstrated by the red peri-nuclear staining. The green UEA staining indicates goblet cell secretory product, denoting the location of goblet cells in the conjunctiva. Arrows indicate goblet cells. The mouse isotype controls (B) were negative as were the rabbit isotype controls (not shown). Epi, epithelium.

Article Snippet: Antibodies used were mouse anti-human NLRP3 (1∶50, Enzo Life Sciences, catalog number 804–819-C100) and rabbit anti-rat caspase 1 (1∶20) (Enzo Life Sciences, Plymouth, PA); rabbit anti-rat ASC (1∶50) (Santa Cruz Biotechnology, CA, USA); anti-rabbit P2X4 and P2X7 (1∶50) (Alomone Labs, Jerusalem, Israel); anti-rabbit TLR2 (1∶100) (Santa Cruz Biotechnology, CA).

Techniques: Immunohistochemistry, Staining, Immunofluorescence, Microscopy

Journal: PLoS ONE

Article Title: Staphylococcus aureus Activates the NLRP3 Inflammasome in Human and Rat Conjunctival Goblet Cells

doi: 10.1371/journal.pone.0074010

Figure Lengend Snippet: Primary cultures of human or rat goblet cells were analyzed by immunofluorescence microscopy after staining with antibodies against inflammasome components NLRP3, caspase 1, and ASC ( A ). All three components were identified as indicated by the red peri-nuclear staining pattern. Rabbit isotype controls (shown) and mouse isotype controls (not shown) were negative. The presence of NLRP3 ( B ), caspase 1 ( C ), ASC ( D ) and IL-1β ( E ) were confirmed by western blot analysis in lysates from human goblet cells and rat conjunctiva and goblet cells. Please note that the positive controls for NLRP3 and ASC are transfected cells lines overexpressing the protein and thus the amount of protein loaded for the western blots was below the detection level of the β-actin antibody. In addition, the NLRP3 is tagged with His tag while the ASC is tagged with a FLAG tag causing these proteins to run at a higher molecular weight than the native molecules.

Article Snippet: Antibodies used were mouse anti-human NLRP3 (1∶50, Enzo Life Sciences, catalog number 804–819-C100) and rabbit anti-rat caspase 1 (1∶20) (Enzo Life Sciences, Plymouth, PA); rabbit anti-rat ASC (1∶50) (Santa Cruz Biotechnology, CA, USA); anti-rabbit P2X4 and P2X7 (1∶50) (Alomone Labs, Jerusalem, Israel); anti-rabbit TLR2 (1∶100) (Santa Cruz Biotechnology, CA).

Techniques: Immunofluorescence, Microscopy, Staining, Western Blot, Transfection, FLAG-tag, Molecular Weight