A IHC IF for PNN and c fos - michelletytong/TRGProtocols GitHub Wiki

Materials & Equipment

  • 800 g NaCl,

  • 20 g KCl,

  • 144 g Na2HPO4 · 2H2O

  • 24 g KH2PO4

  • 8 L of distilled water.

  • Add 15mL Triton X 100 (Final Concentration 0.3%)

  • Add 25g of Bovine Serum Albumin (BSA) (Final w/v concentration 5%)

  • SuperFrost Plus slide (Fisher Scientific 22-034-979)

  • glass dishes/petri dishes to hold brain tissue for mounting

  • coverslips (Fisher Scientific 12544E)

  • Prolong Diamond Antifade Reagent containing DAPI (Thermo Fisher P36962)

  • orbital shaker

  • vortex

  • aluminum foil

  • six-well plates

  • clear nail polish

  • fine paint brushes

  • Alexa 594-conjugated streptavidin (0.67 μg/ml; S11227, Thermo)

  • Alexa 488-conjugated donkey anti-rabbit IgG (1:1,000; A21206, Thermo)

  • Biotin-conjugated WFA (1:3,000; BA-3101, EY laboratories)

  • rabbit polyclonal anti-c-Fos antibody (1:5,000; #226003, Synaptic Systems)

Brief Introduction

Immunohistochemistry (IHC) is a highly useful technique for detection and quantification of target epitopes (e.g proteins, structures, cellular components, etc) in a wide variety tissue types. Applicable to fresh, frozen and FFPE samples, IHC utilizes high specificity antibodies for visualization of protein expression patterns, providing quantitative, qualitative and temporal information on processes taking place in tissues. Antibody-epitope interactions are visualized through secondary staining of samples with an additional antibody that has been conjugated to either a color-producing enzyme (chromogenic staining) or a fluorophore (immunofluorescent staining, or IF).

Immunofluorescent staining (IF) is a subtype of IHC. IF detects specific proteins in tissue sections using primary antibodies to specifically bind to target antigens, which can then be visualized utilizing various fluorescent tags. This technique adds the value of multiplexing, where proteins can be localized within one sample. Additionally, whole slide imaging and analysis is available. (from http://www.mdbiosciences.com/immunohistochemical)

For Summer 2019, we'll be using the indirect Fluorescence method.

Excellent video overview of IHC and Nissl stain (not IF), but greatly shows the free-floating process: https://www.youtube.com/watch?v=M69B7j_usB8

Protocol

Tissue Preparation

Perfusion

  1. Follow steps for Perfusion of Mouse
  2. Post-fix: 4% PFA/PBS 4 degreeC overnight → 30% sucrose/PBS 4 degreeC 2 nights. (Some protocols suggest transferring the brain through graded sucrose solutions, e.g. 15% then 20% then finally 30%, allowing for the brain to sink to the bottom of the jar each time as indication of time to remove. 5 days in each solution is sufficient time for tissue to sink.)

Tissue Sectioning (adapted from http://fluoview.magnet.fsu.edu/applications/protocols/brainfloatingsections.html)

Tissues for Summer 2019: OB, Piriform cortex, Olfactory bulb

  1. See Frozen embedding unfrozen tissue in OCT compound
  2. Cut free-floating, coronal sections of 30 μm using a microtome or vibratome.
  3. Prepare six-well plates filled with PBS.
  4. Transfer one or more frozen (-80 degrees Celsius) floating cryosections to a 6-well culture dish with 1X PBS buffer in each well to allow the sections to thaw and float in the buffer. Slowly bring the culture dish to room temperature and rotate the tissue sections as they are being hydrated on an orbital shaker at 5-10 revolutions per minute.

Note: Do not allow sections to warm to room temperature before adding the 1X PBS buffer. Carefully aspirate all solutions described below away from the floating sections with a Pasteur pipette and a squeeze bulb (do not use a vacuum aspirator).

(Floating sections were fixed before removal from the animal, but can be fixed again after rehydration using paraformaldehyde treatment for 30 minutes. This is an optional step.)

Sample Preparation

  1. Wash each section 4 times in PBS for 15 minutes (each wash). Slowly rotate the tissue sections as they are being washed on an orbital shaker at 5-10 revolutions per minute.

(Optional: Not done for most PNN/c-fos protocols. Dissolve the membranes in the tissue sections with Permeabilization Buffer by treatment for two hours. Slowly rotate the tissue sections as they are being permeabilized on an orbital shaker at 5-10 revolutions per minute.)

Blocking

  1. Block nonspecific secondary antibody and synthetic fluorophore binding sites with 5% BSA/PBS/0.3%Triton X-100 at room temperature (RT or about 20-25degC) for 1 h. Place on orbital shaker at 5-10 revolutions per minute.

Primary Antibody incubation

  1. After blocking, carefully add the primary antibody cocktail without disturbing the tissue sections.

  2. Calculate how much total volume is needed for both primary antibody and negative controls.

  3. Prepare primary antibody solutions: dilute antibody in 1% BSA/PBS/0.3% Triton X-100. [Negative controls TBD.}

  4. Vortex primary antibody; vortex diluent; vortex negative control solutions.

  5. After serum blocking, remember not to rinse.

  6. For PNNs and c-Fos, we will use (Lectin-Reaction) Biotin-conjugated WFA (1:3,000; BA-3101, EY laboratories) and rabbit polyclonal anti-c-Fos antibody (1:5,000; #226003, Synaptic Systems) in 1% BSA/PBS/0.3% Triton X-100 at 4 degree C overnight.

Secondary Antibody incubation

  1. Wash the floating sections 3 times (5 minutes each wash) with PBS. Slowly rotate the tissue sections as they are being washed on an orbital shaker at 5-10 revolutions per minute.

  2. After the wash sequence, prepare secondary anti-body solution.

  3. For PNN and c-Fos, (Biotin-Avidin reaction) Alexa 594-conjugated streptavidin (0.67 μg/ml; S11227, Thermo, ~594 nm, spectra) and Alexa 488-conjugated donkey anti-rabbit IgG (1:1,000; A21206, Thermo, ~488 nm, spectra in 1% BSA/PBS/0.3% Triton X-100 at RT for 90 min.

  4. Wash the floating sections 4 times (10 minutes each wash) with PBS. Cover the culture chamber with aluminum foil (to protect from light) and slowly rotate the tissue sections as they are being washed on an orbital shaker at 5-10 revolutions per minute.

Mounting on slides

  1. Make Mounting Solution: Prolong Diamond Antifade Reagent containing DAPI

  2. Fill a petri dish with 0.5X PBS and transfer the sections to dish.

  3. Tilt the slide (SuperFrost Plus slide) halfway into the dish and use a paint brush to gently move section in place on the slide. (See the video in introduction)

  4. Add a drop of mounting solution (just enough to cover the tissue once the coverslip is applied to the surface.)

  5. Do not let dry - coverslip while still wet. Gently place the coverslip on top of the tissue. Start with one edge against the slide and lower the coverslip gently and slowly, so as not to introduce air bubbles.

  6. Apply a thin layer of nail polish around the perimeter of the coverslip to seal it.

  7. Lay flat to dry at RT, protect from light using dark humidity chamber.

Buffer & Reagent Recipes

Phosphate buffered saline (PBS)

A 10 liter stock of 10x PBS can be prepared by dissolving:

  • 800 g NaCl,
  • 20 g KCl,
  • 144 g Na2HPO4 · 2H2O
  • 24 g KH2PO4
  • 8 L of distilled water.

For 1 liter of 1X PBS, prepare as follows:

  • Start with 800mL of distilled water:
  • Add 8 g of NaCl.
  • Add 0.2 g of KCl.
  • Add 1.44 g of Na2HPO4.
  • Add 0.24 g of KH2PO4.
  • Adjust the pH to 7.4 with HCl.
  • Add distilled water to a total volume of 1 liter.

Post-fix

For 1 litre 30% sucrose/PBS:

  • Start with 1000mL of 1X PBS
  • Add 300g sucrose

For 1 litre 20% sucrose/PBS:

  • Start with 1000mL of 1X PBS
  • Add 200g sucrose

For 1 litre 15% sucrose/PBS:

  • Start with 1000mL of 1X PBS
  • Add 150g sucrose

Blocking Buffer

For 500mL of 5% BSA/PBS/0.3%Triton X-100 Blocking Buffer:

  • Start with 435mL distilled water
  • Add 50mL 10X PBS stock (Final concentration 1X)
  • Add 15mL Triton X 100 (Final Concentration 0.3%)
  • Add 25g of Bovine Serum Albumin (BSA) (Final w/v concentration 5%)

Antibody solutions

For 500mL of 1% BSA/PBS/0.3%Triton X-100 Antibody solution:

  • Start with 435mL distilled water
  • Add 50mL 10X PBS stock (Final concentration 1X)
  • Add 15mL Triton X 100 (Final Concentration 0.3%)
  • Add 5g of Bovine Serum Albumin (BSA) (Final w/v concentration 1%)
  • (Add antibody to the needed dilution, e.g. 1:3000 or 1:5000)

For Summer 2019, use Alexa 488-conjugated streptavidin (comes in 1mg. Add 0.5 mL diH20 to make 2 mg/mL solution. Use this to make a 1:500 dilution in 1% BSA/PBS/0.3%TritonX-100 antibody solution)