Crystal Immunostaining

Note: Inspired by Katie Goodwin, Crystal Rogers, and Eric Fowler, we use TBS with \(CaCl_2\) to optimize ECad staining in the chicken. Gluteraldehyde doping can help perserve Mitochondria, and Isopropanol is to accomidate Phalloidin. If you can drop Phalloidin, Methanol is better.

Room Temperature
- TBS: TBS without \(CaCl_2\)
- TBS-C: TBS with 0.0735 mg/mL \(CaCl_2\) (0.66 mM)
- TBST-C: TBS-C and 0.5% Triton (or 0.1% Triton for fragile stains such as pMLC and aSMA).



On Ice or 4°C
- PFA: 4% PFA diluted into PBS
- Donkey Block: 10% Donkey Serum with TBST-C
- Alt: Block-Aid: 1:1 with TBST-C
- Primaries Primary Antibodies for Day 1
- Secondaries and Stains Secondary Antibodies and Stains for Day 3





Day 1

  1. Fixation: Add sample to cold PFA with at least a 1:20 sample-to-PFA volume ratio.
    • Use 4% just-made or just-thawed cold PFA in PBS.
    • Use 1 mL PFA by default.
    • Note this process will greatly reduce any pre-fixed fluoressence signal that your sample might have.
  2. Place on a cold shaker (4°C) for 30 minutes to allow PFA to diffuse. 30:00
    • PFA diffusion into the lung at 4°C takes ~30 minutes for 400 µm.
    • PFA crosslinking is endothermic and requires heat. So this is slowed down at 4°C compared to RT.
    • Over-crosslinking blocks target antigen sites!
  3. Optional: For large samples, bring the sample back to room temperature (RT) to allow PFA to cross-link for 15 minutes. 15:00

TBS and Calcium?

TBS is not used for the PFA step, because Tris has an amine group. After that however, the phosphate groups in PBS can interact with and modify proteins such as pMLC, while Tris is relatively inert.

The extracellular portion of E-cadherin has repeating domains where Ca binds to create a rigid conformation, making Ecad recognizable to the primary antibody. This protocol uses the lower end of physiological Ca levels.

Formaldehyde Chemistry

\(CH_2O\) reacts with primary amine groups, \(R-NH_2\), in proteins. This forms \(R-N=CH_2\) which can react with another amine group to form a stable methylene bridge \(R-NH-CH_2-NH-R\\'\). These bridges cross-link proteins, solidifying the biological structure.

Gluteraldehyde Chemistry

\(CHO-(CH_2)_3-CHO\) is a dialdehyde, it has two aldehyde groups and creates two methylene brideges per molecule. The delicate structure of mitochondria get destroyed over time by the floppy Formaldehyde fixation, but the Gluteraldhyde reinforces the fixation keeping the crisp structure of mitochondria.

  1. Wash out fixative Transfer from PFA to 500 µL PBS, leave for 15 minutes at RT on a shaker. 15:00
    • PBS at this step to minimize Tris-PFA interactions.
  2. Permeabilize and wash Wash 3 times with 500 µL 0.5% TBST-C for 15 minutes each at RT on a shaker. 15:00
    • Use 0.1% TBST-C for some weaker-bound antibodies like αSMA.
    • TBST-C washes help clear the sample.
  3. Block Inncubate with blocking solution for 1 hour at RT on a shaker. 60:00
    • Use 500 µL 10% Donkey Serum in TBST.
    • Alt, use BlockAid 1:1 with TBST
  4. Primary Antibody Incubation: Combine primary antibodies with blocking solution and mix gently.
    • Note any volume alterations.
    • Do not vortex! Vortexing oxyginates the liquid.
    • Incubate 48 hours at 4°C on a shaker; larger samples may require longer.

Day 3

  1. Wash out unbound primary: Wash 4 times with 500 µL TBST-C for 15 minutes each at RT on a shaker. 15:00
  2. Re-Block: Block with 500 µL for 1 hour at RT on a shaker. 60:00
  3. Secondary Antibody Incubation: Combine secondaries with blocking solution and mix gently.
    • Add non-antibody dyes (DAPI, Phalloidin) and protect from light as needed.
    • Centrifuge secondaries before use; incubate 24 hours at 4°C on a shaker.

Day 4 - Washing & Clearing

At this point, it is actually good to get the salts off. So ideally you now use TBS without CaCl, or at least use PBS. Salts can precipitate out in the alcohol solutions.

  1. Wash out unbound secondary: Perform 1 wash with TBST (No CaCl!) for 15 minutes, 15:00
    • Then wash in TBS (No CaCl!) for 15 minutes. 15:00

  2. Dehydrate/Clear: Titrate through Isopropanol (or Methanol) series for 15 min each, keeping in TBS (No CaCl!).

    • 25% Isopropanol 15:00
    • 50% Isopropanol 15:00
    • 75% Isopropanol 15:00
    • 100% Isopropanol 15:00
    • 100% BABB

  3. Place coverslip onto washer, There are two ways to do this: - Add BA:BB until it is forms a dome above the nylong washer, then drop a glass coverslip directly onto it. - Or, slide a glass coverslip onto the sample from one side, while pipetting extra BA:BB from the other side to avoid air bubbles.

  4. Image promptly, within the week. DAPI fades and bubbles form over time.

References:
- Plasticity in airway smooth muscle differentiation during mouse lung development
- Comparative analysis of fixation techniques for signal detection in avian embryos

Solved Questions

Open Questions

  • Can the detergent (Triton) concentration be lowered? Does it improve the image?
  • Is another detergent such as Saponin better for imaging mitochondria?
  • Does a gradual increase in PFA concentration work better?
  • How much better is the signal preserved by a secondary round of PFA after the primary antibodies?
  • Gluteraldehyde has started given mixed results, what is the best gluteraldehyde protocol?