Table 1.
Primary antibodies used in this research.
Table 2.
Secondary/tertiary antibodies used in this research.
Figure 1.
Immunohistological analysis of thymic epithelial cell-associated molecules in the thymi of rats and mice.
Double immunoenzyme staining for molecules of interests (blue) and tissue frameworks (type IV collagen, brown) Cortical (subcapsular) and medullary epithelium-free areas unique to rats are indicated by “cEFA” and “mEFA”, respectively. Epithelium-containing areas in the medulla are indicated by “mECA”. The antibodies used were anti-K5 followed by anti-rabbit IgG, anti-K8 followed by anti-chicken IgY, and anti-mouse CD205 followed by anti-rat IgG. Anti-rat MHCII and anti-rat CD205 followed by biotin-conjugated anti-mouse IgG, and biotin-conjugated anti-mouse MHCII, and biotin-conjugated ED18/ED19/ED21 were further reacted with anti-biotin antibody. Secondary antibodies and anti-biotin antibody were all conjugated with alkaline phosphatase and developed with the Vector Blue substrate kit. Tissue frameworks were stained with anti-type IV collagen antibody followed by peroxidase-conjugated anti-rabbit IgG and developed with 3, 3′-diaminobenzidine substrate.
Table 3.
Expression of TEC-related markers in thymi of rats and mice.
Figure 2.
Characterization of cortical thymic epithelial cells.
Frozen sections of rat (A-C), and mouse (E) thymus, and a cytosmear from isolated rat thymic nurse cells (D) were subjected to multicolor immunofluorescence staining. The following antibodies were used: Alexa488-conjugated ED18, Alexa488-conjugated ED19, anti-rat CD45 antibody followed by Alexa594-conjugated anti-mouse IgG antibody, anti-K5 antibody followed by AMCA-conjugated anti-rabbit IgG antibody, anti-K8 antibody followed by Alexa-594 conjugated anti-chicken IgY antibody, Alexa647-conjugated anti-rat MHCII antibody and Alexa647-conjugated anti-mouse MHCII antibody.
Table 4.
Phenotype of TEC subsets in rats and mice.
Figure 3.
Characterization of rat medullary thymic epithelial cells 1: relationship between ED18 and ED21 staining.
A section of thymus from a Lewis rat was stained with Alexa594-conjugated ED18, Alexa488-conjugated ED21, and Alexa647-conjugated anti-rat MHCII antibody. Pseudocolors were adjusted and assigned with AxioVision software. Merged images are explained schematically below the panels.
Figure 4.
Characterization of rat medullary thymic epithelial cells 2: relationship between keratin expression and ED18/ED21.
Sections of thymus from a Lewis rat were stained with Alexa488-conjugated ED18 (A, B) or Alexa488-conjugated ED21 (C). Subsequently, the sections were stained with Alexa647-conjugated anti-rat MHCII antibody, anti-K5 and anti-K8 followed by AMCA-conjugated anti-rabbit IgG and Alexa594-conjugated anti-chicken IgY antibodies, respectively. Pseudocolors were adjusted and assigned with AxioVision software. Pictures in (A) and (B) are from the same multicolor picture with different color assignments. Merged images are explained schematically below the panels.
Figure 5.
Characterization of rat medullary thymic epithelial cells 3: UEA-1 binding to mTEC subpopulations.
(A) A section of a thymus from a Lewis rat was stained with Alexa594-conjugated ED18 or ED21, Alexa647-conjugated anti-rat MHCII antibody, and biotin-conjugated UEA-1 followed by Alexa488-conjugated anti-biotin antibody. Pseudocolors were assigned using AxioVision software. (B) The low-density fraction from Lewis rat thymi was subjected to flow cytometric analysis. Cells were stained with Alexa488-conjugated control IgM or ED18/ED21, anti-rat CD45-PE, biotin-conjugated UEA-1 followed by streptavidin-PerCP-Cy5.5, and Alexa647-conjugated anti-rat MHCII.
Figure 6.
Expression of functional molecules in rat mTEC1 and mTEC2 subsets.
(A) Rat thymic sections were stained with Alexa488-conjugated ED18 or ED21 (green) and anti-AIRE antibody followed by Alexa594-conjugated anti-goat IgG (red). Tissue framework was stained blue with anti-type IV collagen followed by AMCA-conjugated anti-rabbit IgG. Arrows indicate AIRE expression associated with ED18- and ED21-positive cells. (B, C) Rat thymic sections were stained with anti-claudin-3 or anti-claudin-4 antibodies followed by AMCA-conjugated anti-rabbit IgG, Alexa488-conjugated ED18, Alexa594-conjugated ED21, and Alexa647-conjugated anti-rat MHCII. Pseudocolors were assigned using AxioVision software.
Figure 7.
Epithelium-free areas in the rat thymic medullas.
(A) Frozen sections of Lewis rat thymi were stained with anti-CD205 or anti-CD103 antibodies followed by biotin-conjugated anti-mouse IgG antibody and Alexa488-conjugated anti-biotin antibody, Alexa-594 conjugated ED18 antibody, Alexa-647 conjugated anti-rat MHCII antibody, and anti-type IV collagen antibody followed by AMCA-conjugated anti-rabbit IgG antibody. Pseudocolors were assigned using AxioVision software. (B) Whole images of ED21- stained thymic sections from three Lewis rats were captured, and mECAs and mEFAs were calculated with cellSense software.