RNA Detection

2.4 Antibody Based
Rapid Tissue Staining

1. Aluminum foil to cover light-sensitive staining solutions.


2. 15 mL centrifuge tubes.


3. Staining containers with 25 mL of ddH 2 O and PBS (replaced
   with fresh liquids for every wash).


4. Staining containers for dehydration: 150% ethanol, 175%
   ethanol, 195% ethanol and 199.7% ethanol, 25 mL each.


5. Shaker.


6. Acetone, ice cold in a staining container (25 mL per staining).


7. Freshly made 0.25% Triton X-100 in PBS to dilute primary and
   secondary antibodies (500μL per slide).


8. Primary antibody (volume is dependent on the antibody con-
   centration needed), kept on ice until diluted for use.


9. Secondary antibody (the volume is dependent on the antibody
   concentration needed), kept on ice until diluted for use.


10. 2.5 mL of streptavidin–biotin solution (ABC solution, e.g.,
    Vector Laboratories Vectastain ABC kit Elite) in a 15 mL
    tube (covered with foil and kept in motion for 30 min prior
    to use).


11. 2.5 mL of diaminobenzidine solution (DAB solution, e.g.,
    Vector Laboratories DAB Kit) in a 15 mL tube (covered with
    foil and kept in motion for 2–3 min prior to use).


12. 1000μL pipette and filter tips.

2.5 Library
Preparation

1. Thermocycler, if possible dedicated to this procedure.


2. Iron thermal block stand.


3. Styrofoam box with ice.


4. Vortex.


5. Microcentrifuge.


6. Pipettes and filter tips for different volumes.


7. Timer.


8. Nuclease-free 1.5 mL tubes for preparation of master mixes.


9. PCR tubes.


10. Nuclease-free ddH 2 O.


11. PEG (polyethylene glycol) 8000.

2.5.1 Reverse
Transcription and PCR
Amplification

1. Reverse transcription kit (e.g., Invitrogen SSRT II).


2. ERCC spike ins (2.5 105 dilution in ddH 2 O, prepare ali-
   quots in a sterile laminar flow hood and store at
   80 C, do not
   freeze-thaw).


3. dNTPs mix (10 mM each nucleotide).

98 Susanne Nichterwitz et al.