In situ hybridization technique

SLIDE PREPARATION FOR IN SITU HYBRIDIZATIONS

WEAR GLOVES! WEAR GLOVES! WEAR GLOVES! WEAR GLOVES!

  • Your hands are a good source of RNase, avoid touching any of the slides or slide boats with bare hands throughout all steps.
  • Use slide boats and dishes that are designated for in situs.
  • Acid clean the slides.
  • Be sure to rinse in tap water and running distilled water.
  • Avoid any contamination with general labware.

Subbing
DEPC water is defined as milli-Q water mixed vigorously with 0.1% Diethylpyrocarbonate for 2 hours and then autoclaved.

Heat 500 ml of DEPC water to 50ºC, do not let it boil! Add 5g of gelatin while mixing the water with a stir bar.

Place the flask in a bucket with ice and cool to about 40ºC. Add 0.5g of chromium potassium sulfate (chrome alum). Use warm. Avoid creating bubbles when dipping the slides in the subbing solution. Cover and let dry overnight.

Poly-l-lysine coating
(Remember you are wearing gloves!!)

Dissolve 0.60g of Tris base in 500 ml of DEPC water (to make 10 mM) and adjust the pH of the buffer to 8.0.

Dissolve 25 mg of poly-L-lysine in 500 ml of the 10 mM Tris buffer.

Dip the subbed slides in the poly-L-lysine solution for 10-30 min. Let them dry overnight in a 37-50ºC oven.

When the slides are dry store them in the slide boxes dedicated for in situ hybridization.

MATERIALS AND METHODS FOR IN SITU HYBRIDIZATIONS

Barthel, L. K. And Raymond, P. A. (1993), Subcellular localization of tubulin and opsin mRNA in the goldfish retina using digoxigenin-labeled cRNA probes detected by alkaline phosphatase and HRP histochemistry. J. Neurosci. Methods, 50: 145-152.

HYBRIDIZATION PRE-TREATMENT

(The solutions are made according to protocols found in the Genius System Users’s Guide for Membrane Hybridization, Version 3.0 from Boehringer Mannheim)

All reagents should be stored at 4ºC or -20ºC.

All glassware and labware is oven baked overnight at 210ºC or autoclaved.

DEPC water is defined as milli-Q water mixed vigorously with 0.1% Diethylpyrocarbonate for 2 hrs. and then autoclaved.

Sections are cut using a brush dedicated for in situ and gloves are worn. After sectioning the slides are placed in avacuum desiccator for a minimum of 2 hours or overnight. The slides are then store with desiccant in the -90 ºC freezer. When slides are removed for use care is taken to avoid warming the remaining slides thus preventing the condensation of moisture on them.

Remove formamide aliquot from freezer.

20X SSC:

  • Dissolve 87.6g of NaCl in 350 ml of milli-Q water
  • Add 44.12g Sodium citrate bring to a final volume of 500 ml with milli-Q water

Hydrate the slides in ethanol series, 100% 2X, 1X each 95%, 70%, 50%, and finally 2X SSC, 1 min. each.

Proteinase K Buffer: 0.1M Tris pH 8.0 50 mM EDTA

  • 25 ml 1M Tris
  • 25 ml 0.5M EDTA (pH 8.0) bring solution up to 250 ml with DEPC water

Prewarm 250 ml Proteinase K and buffer to 37ºC. Add 250 ?l of Proteinase K [10 mg/ml] to the buffer just before use.

Incubate the slides in the Proteinase K solution. This is tissue and probe dependant. A time course series should be done to determine optimal signal localization. Too short, detection is sterically inhibited, too long, signal is lost. 5 minutes for 10 ?m goldfish retinas is the max.

Rinse briefly in RT DEPC water.

TEA needs to be made fresh every time.

  • 9.3g in 490 ml DEPC water and pH to 8.0 using NaOH pellets.

Rinse in 0.1 M TEA, pH 8.0, for 3 minutes. Add 650 ?l Acetic anhydride to a dry dish and pour in the remaining 250 ml TEA. Incubate the slides in this solution for 10 minutes.

Dehydrate the slides in the reverse order of the steps used in hydration. Drain the slides and let them dry at RT for at least 1 hr.

HYBRIDIZATION

Boil the probe for 10 min and then quickly cool on ice, add hybridization solution components to the tube containing the probe.

Hybridization solution:

  • 36 ?l TEN buffer
  • 250 ?l 100% D.I. Formamide
  • 100 ?l 50% Dextran Sulfate (50% in DEPC water)
  • 50 ?l 10% Blocking soln.
  • 2 ?g DIG RNA probe (=X ul)
  • 64 ?l – X ?l (vol of probe) DEPC water

TEN Buffer:

  • 5 ml 1.0M Tris-HCl, pH 7.5
  • 30 ml 5M NaCl
  • 1 ml 0.5M EDTA

Place enough probe solution on the coverslip to completely cover the sections (approximately 60 ?l). Gently lower the slide to the RNase free and siliconized coverslip.

Seal the edges with DPX and incubate in humid chamber at 56ºC overnight.

Alternatively:
Grace Bio-Labs (Sunriver OR, gracebio@teleport.com) manufactures RNase free siliconized HybridSlips. We use the 22 x 60 mm size (cat. #HS60) with 75 ?l of hybridization solution. The coverslips are not sealed and the hybridization is done in a humid chamber.

IN SITU POST HYBRIDIZATION

Formamide solution:

  • 50% molecular grade formamide in 2X SSC.

Do not discard this wash, it is reusable. Preheat the formamide wash to 65ºC.

RNase Buffer: 0.5M NaCl, 10 mM Tris, pH 7.5, 1 mM EDTA

  • 29.23g NaCl
  • 10 ml 1M Tris pH 7.5
  • 2 ml 0.5M EDTA
  • bring up to 1L with DEPC water

Preheat two RNase buffer washes without the RNase, one 200 ml to 37ºC, and the second to 65ºC.

Pre heat two 2X SSC washes to 37ºC

The following washes are at room temp. on the shaker table:

Remove the DPX and soak the slides in 2X SSC for 30 minutes. If the coverslips have not already floated off, leave the slides in the buffer and gently tease the cover slip off.

If the hybridization was done using the HybridSlips gently slide the coverslips off and wash the slides in 2X SSC for 30 minutes then continue with the 65? C formamide wash.

Wash an additional 30 minutes in fresh 2X SSC.

Rinse in the preheated formamide solution for 1/2 hr. Agitate slightly for the first 5 minutes.

Rinse the formamide in 2X SSC at 37ºC, two times 10 min. each.

Add 400 ?l RNase A [10 mg/ml] to the 200 ml 37ºC RNase buffer and incubate the slides in the solution for 30 min.

Wash the slides in the 65ºC RNase buffer (without RNase), for 30 minutes.

Immunohistochemical detection

Wash 2-3 hours in 2X SSC + 0.05% Triton X-100 + 2% Blocking reagent, (use 10% blocking stock, made from Genius blocking reagent.)

5X Maleate buffer:

  • 58g Maleic acid in 850 ml milli-Q water pH to 7.5 using lots of NaOH pellets
  • Add 43.8g NaCl bring up to 1L with milli-Q water (dilute to 1X for working concentration)

Wash in Maleate buffer, 2X 5 min. each.

Carefully dry a ring around the sections without allowing the sections to dry and draw on a PAP pen ring.

Incubate slides in humid chamber 3-5 hr or overnight at room temp. with apprx. 200-500 ?l DIG-Alkaline Phosphatase antibody, diluted 1:1000-1:3000 in Maleate buffer + 1% Blocking reagent + 0.3% Triton. (DIG-AP Ab. is stored in the dark at 4ºC.)

Alternatively:
Grace Bio-Labs supplies water tight chamber slips, CoverWell, 200 ?l vol. (cat. #PC200). The slide is lowered onto the CoverWell containing 205 ?l antisera.

Wash in Maleate buffer 2X 10 min. each.

Wash in Genius kit 3 buffer 3 1X 10 min.

Buffer 3:

  • Mix 100 ml of 10X Tris/NaCl stock in 800 ml milli-Q water check the pH at this time, it should be around 9.5
  • While mixing add 100 ml of the 10X MgCl2 stock
    *****do not adjust the pH at this point*****

10X Tris/NaCl stock: 1M Tris HCl + 1M NaCl

  • In 350 ml milli-Q water add: 78.5g TrisHCl let dissolve then add:
  • 29g NaCl.
  • Use lots of NaOH pellets to pH to 9.5, bring to vol. 500 ml, sterile filter using bottle top filter on a very clean bottle.

10X MgCl2 stock:

  • Dissolve 50.8g MgCl2 in 420 ml of milli-Q water
  • Bring up to 500ml and sterile filter into a very clean bottle.

Incubate in freshly prepared chromogen substrate prepared in buffer 3. Plate 200-500 ?l per slide.

Grace Bio-Labs CoverWells can be used for chromogen substrate incubation. After placing the CoverWell on the slide place them upside down in the humid chamber. If any precipitate should form during the color reaction it will fall on the CoverWell and not on the section.

Chromogen substrate:

  • 45 ?l NBT-solution (Genius kit 3, vial 4)
  • 35 ?l X-phosphate soln (Genius kit 3, vial 5)
  • 10 ml buffer 3.
    or
  • NET/BCIP ready-to-use tablets from BMB catalog # 1697-471

Incubations are dependant on the strength of the signal. Increasing the temp. to 37ºC only accelerates the reaction, it will not intensify it, and the nonspecific background becomes a problem. Time course for the incubations can run from a couple hours to overnight. Be sure to use a very humid chamber to prevent the slides from drying.

Alkaline phosphatase substrate wash:

  • Dissolve 1.75g Tris-HCL in 950 ml milli-Q water pH to 7.5, bring to IL.

After color reaction is complete wash the slides in the Alkaline phosphatase substrate wash for at least 30 min. Sections are mounted under coverslips with glycerol. Seal the edges of the coverslips with Permount.

Things to keep in mind:

The work space must be clean, all glassware is baked at 210ºC for at least 24 hrs, and plasticware is DEPC treated and autoclaved prior to use. After the initial DEPC treatment only autoclave the plasticware. Periodically DEPC treat the plasticware again, especially when there is a problem with the in situs. Gloves are worn at all times during prehybridization steps.

Proteinase K digestion may vary with probes and tissue. It may be wise to do a series of time points to determine the optimal length of digestion. 30 seconds – 5 minutes would be a good start.

Signal detection is dependant on the concentration of probe in the hybridization solution. For most purposes 4 ng/?l should work when 60 ul is plated on the slide, (2 ?g probe/500 ?l hybdz. soln.). Some probes used have given strong signals at 1 ng/?l, others can only be detected at a concentration of at least 4 ng/?l.

The best way to store the probe is to aliquot it into working concentrations in Rnase free and siliconized tubes, and keep frozen at -20ºC.

Leftover hybdz. solution that contains the probe can be stored at -20ºC and used again within 2 weeks or more, it must be boiled again before use.

If you get negative results first try repeating the immunocytochemistry, be sure that all your buffers are at the correct pH. The alkaline phosphatase reaction is especially sensitive to the pH of the buffer.