ADS Immunoflourescence

ADS IF protocol from Jenn

Blocking

 * Outline with hydrophobil pen
 * Wash slides with cold MeOH or MeOH:Acetone (1:1 in freezer) for 6-10 min in glass containers (can put slides back to back)
 * Lay out slides on double serological pipets—do not let dry out
 * Block with 1:20 horse serum (in freezer by Shiying) in PBS for 20 min (200-400µl/slide)
 * Remove by dumping onto paper towel

Primary Ab

 * Dilute Ab in PBS with horse serum (1:50)—add ~400ul per slide
 * Incubate 30 min-1hr
 * Wash 4X in PBS (5min)

Secondary Ab (IN DARK)

 * AF 488(anti-mouse for ColVII) or AF 555 (anti-rabbit for KRTs) 10ul each, 39ul horse serum, 3900ul PBS (In Shiying’s box) (1:500 dilution)
 * Incubate 30 min-1hr
 * Wash 4X in PBS (5 min)
 * Add Hoescht for 5 min
 * Wash 2X in PBS (5 min)

Antibodies Used for ADS (all at -20º unless noted):

 * Basal: Integrin B1(BD Pharmigen, Rat, 4º), K5 (Covance, Rabbit), K14 Covance, Rabbit) NOTE: K5/K14 stain all cells in ADS
 * Spinous: K1 (Covance, Rabbit), K10 (DAKO Clone DE, Rabbit)
 * Granular/Stratum Corneum: Loricrin (Covance, Rabbit)/Involucrin (Covance, Rabbit)/Transglutaminase (Mouse, 4º)
 * BMZ: Collagen 7 (Chemicon MAB 2500, Mouse, 4º; Clonetech, Rabbit)

Combinations:

 * Basal/BMZ: K5 (Rb 1:500) + Col 7 (Ms 1:200)
 * Spinous/Basal: K1 (Rb 1:200) + Col 7 or Int B1 (Rat 1:100)
 * Spinous/BMZ: K10 (Ms 1:200) + Col 7 (Rb 1:200)
 * Granular/BMZ: Loricrin (Rb 1:500) + Col 7 (Ms 1:250)