Immunostaining

For my Immunostaining Protocol, see here

What is Immunostaining?

When you are staining, you are working on getting molecules, both small and large, to interact with the tissue as uniformly as possible. While working, you can think of these processes as reaction-diffusion processes. Here are my thoughts on the molecular dynamics of the four major processes:

  • Fixation
  • Staining through antibodies
  • Staining through small molecules
  • Clearing

Fixation

Formaldehyde \(CH_2O\) 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 \(CHO-(CH_2)_3-CHO\) 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.

To Do: Put some illustrations of what fixation looks at a molecular scale like here To Do: Put the diagrams of the chemicals down.

A rule of thumb is that for every 10°C rise in temperature, the rate of reaction doubles. So for fixing large samples, I try to spread the fixative out as much as possible at very cold temperatures, and then bring the sample up to room temperature for even fixation.

Diffusion of the fixative

Formaldehyde has a molecular weight of approximately 0.03 kDa or 30 g/mol. Its effective radius as a small molecule is roughly 0.1 nm (or 1 Angstrom). So at room temperature (20°C), formaldehyde diffuses at approximately 1000 µm²/s.

You can try to calculate diffusion coefficients \(D\) from the particle’s radius \(R\) and the media’s viscosity \(\eta\) through the Stokes-Einstein equation:

\[ D= \frac{k_B T}{6 \pi \eta R} \]

But because you are diffusing through tissue, it’s going to be slower.

Diffusion through tissue will occur at a speed more similar to 10-100 µm²/s, unless the tissue has some particularly difficult structure to get through like in muscle. The wide range depends heavily on tissue density and composition.

So if we ignore convection, how much time does it take PFA (formaldehyde) to diffuse through 100 µm of tissue at 4°C?

The characteristic time (\(t\)) for diffusion across a distance (\(d\)) can be estimated using the equation:

\[ t \approx \frac{d^2}{2D} \]

For 4°C (277.15 K), assuming \(D \approx 50 \text{ µm}^2/\text{s}\):

\[t \approx \frac{(100 \text{ µm})^2}{2 \times 50 \text{ µm}^2/\text{s}} = 100 \text{ seconds}\]

How much time does it take at 20°C (293.15 K)? Since \(D \propto T\), and \(t \propto 1/D\), logically the time to diffuse a distance goes as the inverse of the temperature. Double the temperature, halve the time. But note temperature is in Kelvin, so changing from 4°C to 20°C is not such a large difference.

Note the \(d^2\). Diffusing through a large sample can take a long time.

To help speed this whol process up, we put the sample in a rocker to swish the fluid all around. At very least the PFA will be moving through the tissue from every nook and cranny. The penetration depth we care about then is measured from all the surfaces available

Antibodies

Primary Antibodies

Secondary Antibodies

Diffusion of Primary and Secondary Antibodies

Antibodies are much larger than chemicals. They are ~150 kDa with a radius of ~5 nm. Their diffusion constants in aqueous solution are ~50 µm²/s, but in dense tissue this is more like 1 µm²/s.

So now to diffuse 100 µm, it will take:

\[ t \approx \frac{(100 \text{ µm})^2}{2 \times 1 \text{ µm}^2/\text{s}} = 1.4 \text{ hours} \]

Much longer. This is where the problems start to arise. Again, this time increases with the square of the distance, so it gets rough very fast! If your sample is getting darker towards the middle, there is a good chance your primaries/secondaries didn’t make it all the way into the tissue.

Stains

Clearing

To Do:

Some things I need to put here eventually

  • Debugging tips for too much/ too little primary/secondary/fixation
  • How to select antibodies online
  • Gluteraldehyde, different fixaition methods
  • Clearing methods and photophysics of clearing.