Neuron Recovery of Neurobiotin/biocytin cells¶
Contributed by Luke Hammond, QBI, The University of Queensland, Australia
Neuron recovery of neurobiotin/biocytin filled neurons. The method may applied to free-floating or cryostat sectioned fixed tissue.
This technique results in a black insoluble reaction product delineating the filled neuron. Developing the chromagen is slow and should be monitored microscopically. The reaction is stopped before the background staining occurs. The labeled sections can then be counter stained and mounted permanently.
DAB is a carcinogen and all powder must be weighed in a fume hood with appropriate PPE. Disposal and deactivation of excess powder and contaminated washings with dilute bleach solution prior to disposal into the sink is mandatory. Nickel Sulfate must be disposed of appropriately.
24 well plate Sable brush 1XPBS buffer PBS Tween Buffer: 0.05% Tween in 1XPBS Acetate buffer: 0.1M Sodium acetate trihydrate bring to pH to 6.0 with Glacial Acetic Acid NiSO4 solution(0.13M) in Acetate buffer (40g Ni SO4 in 2L of acetate buffer adjust to pH 6.0 with HCl) – Note: NaOH will produce a precipitate. D-glucose Ammonium Chloride Glucose Oxidase 30% H2O2 Fetal calf serum ABC vector elite kit PK6100 NiSO4/DAB solution (dissolve DAB in approximately 250?l of water before adding to NiSO4/acetate buffer)
- Collect sections into multi well plate using sable-hair brush.
- Wash sections well in 3 x 5min of PBS-Tween
- Oxidise sections in PBS- H2O2 (750?l of 30% H2O2 in 25ml PBS) for 30min (this step quenches the endogenous peroxidase activity in the tissue.)
- Wash sections well in 5 x 5min of PBS.
- Incubate sections in blocking solution (25ml PBS + 0.5ml fetal calf serum) for 1 hour (this step blocks non-specific protein interactions.)
- During this time make ABC solution (6?l of part “A” and 6?l of part “B” per ml of solution required) or as stipulated by the manufacturer. Add total amount of A and B to 1/5 of the final volume of PBS and stir at room temp for at least 30min. Make up to the final volume with the remaining PBS just prior to use.
- Sections are incubated overnight in ABC solution at 4°C on a shaker.
- Wash sections well in 3 x 10min of PBS
- Wash sections 5min in acetate buffer
- Pre-incubate sections for 15min in NiSO4/DAB solution without glucose oxidase.
- Incubate sections in fresh NiSO4/DAB solution with glucose oxidase. Reaction can take up to 30min.
- To stop the reaction, wash quickly with acetate buffer.
- Wash again in acetate buffer for 5min to prevent background staining.
- Wash 3x5min PBS. An overnight wash can help reduce the background even further.
- Mount free floating sections onto chrome alum gelatin coated slides and dry.
- Counterstain, dehydrate, clear and mount in DPX.
Smith DW, Day TA Neurochemical identification of fos-positive neurons using two-colour immunoperoxidase staining. J Neurosci Methods (1993) pmid:8100600