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This antibody product recognizes human CCR5.

100 μl of IgG solution in PBS with 50% glycerol. Store at -20 C.

Key points: 1:200 dilution in antibody diluent; 30 min incubation. 

Agilent/Dako EnVision+ Dual Link (HRP-polymer) secondary reagent is required for optimal results.

Immunohisto/cytochemistry protocol (to be adapted by user):

A. Deparaffinize and hydrate sections:

Heat the sections at 58°C for 25 minutes. Incubate sections in three washes of xylene for 3 minutes each. Incubate sections in two washes of 100 % ethanol for 2 minutes each. Incubate sections in one wash of 95 % ethanol for 1 minute. Incubate sections in one wash of 70 % ethanol for 1 minute. Wash sections with running tap water for 1 minute. Wash Sections with DI water for 1 minute.

B. Antigen Retrieval (Performed in DAKO PTLink)

Heat sections in 10 mM sodium citrate buffer (pH 6.0) at 97 ℃ for 10 minutes in DAKO PTLink. Cool slides for 20 minutes after antigen retrieval.

C. Immunohistochemical Labeling. (Steps below are performed in DAKO Autostainer Plus)

Wash sections with 1X TBST. Incubate sections in 3% hydrogen peroxide at room temperature for 10 minutes. Wash sections with 1X TBST. Block section with 200μl/zone 10% goat serum in Antibody Diluent (Invitrogen) at room temperature for 30 minutes. Blow off blocking solution and block each section with 200μl/zone Avidin Blocking solution at room temperature for 15 minutes. Wash sections with 1X TBST. Block each section with 200μl/zone Avidin Blocking solution at room temperature for 15 minutes. Wash sections with 1X TBST. Add 200μl/zone diluted primary antibody to each section. (Dilute AX100 antibody 1:200 in Antibody Diluent with background reduction) incubate at room temperature for 30 minutes.

Wash sections in 1X TBS three times. Add 200μl/zone diluted secondary antibody (. EnVision+ Dual Link, Single Reagents (HRP. Rabbit/Mouse). to each section. Incubate at room temperature for 30 minutes. Wash sections in 1X TBST three times. Add 200μl/zone DAB reagent to each section and incubate at room temperature for 3 minutes.  Wash the sections with DI water twice. 

D. Counterstain, dehydrate, clear. Wash sections with running tap water for 1 min. Counterstain sections in CAT hematoxylin 15 seconds. Wash sections with running tap water for 1 min. Differentiate in Clarifier 1(Thermo scientific/Richard-Allan Scientific). Wash sections with running tap water for 1 min. Bluing in Bluing Reagent (Thermo scientific/Richard-Allan Scientific) for 1 minute. Wash sections with running tap water for 1 minute. Dehydrate from 70% alcohol to 95% alcohol, 3 changes of absolute alcohol, 1 minute each. Clear in 3 changes of xylene Substitute (Thermo scientific/Richard-Allan Scientific), 1 minute each.

E. Coverslip