# How do people accurately and methodically draw laminar boundaries in spinal cord IHC images? **URL:** \ **Category:** Microscopy (IHC/ISH/ICC/In Vivo)\ **Created:** [October 31, 2017, 4:52pm UTC](https://forum.painresearcher.net/t/how-do-people-accurately-and-methodically-draw-laminar-boundaries-in-spinal-cord-ihc-images/318 "2017-10-31T16:52:19Z")\ **Posts on this page:** 9\ **Page:** 1
**Author:** ![achamess](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/achamess/32/534_2.png) [@achamess](https://forum.painresearcher.net/u/achamess)\ **Post date:** [October 31, 2017, 4:52pm UTC](https://forum.painresearcher.net/t/how-do-people-accurately-and-methodically-draw-laminar-boundaries-in-spinal-cord-ihc-images/318/1 "2017-10-31T16:52:19Z")
Many papers that do spinal IHC in the dorsal horn superimpose boundaries on the laminae. ![27FF2](https://canada1.discourse-cdn.com/flex030/uploads/painresearcher/original/1X/b1a020819fbcd494f0952e5b2a7eea2ac5740da2.png) ([http://anesthesiology.pubs.asahq.org/article.aspx?articleid=1944809](http://anesthesiology.pubs.asahq.org/article.aspx?articleid=1944809)) How do people consistently and accurately do this? What are the features or landmarks that you use? I know that certain molecular markers define laminar areas, for example PKC-gamma and lamina II inner and IB4 as lamina II outer. But in the absence of these markers, how do people do it? Neuronal density? that means you need to always use NeuN or Nissl. But that then takes up a color channel. What do you do? And what methods are there in the literature? @cedric.peirs@tberta @zhzhj131421 @esypek @liz @janehartung ---
**Author:** ![liz](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/liz/32/267_2.png) [@liz](https://forum.painresearcher.net/u/liz)\ **Post date:** [October 31, 2017, 6:37pm UTC](https://forum.painresearcher.net/t/how-do-people-accurately-and-methodically-draw-laminar-boundaries-in-spinal-cord-ihc-images/318/2 "2017-10-31T18:37:30Z")
What I usually do is collect a couple sections from the same animal into a separate well, or onto a separate slide, for Nissl staining. Then I can apply boundary lines to the Nissl sections and measure the approximate distances between lines (or from the gray matter border, in the case of lamina I) in image processing software, then apply those measurements to my IHC-stained sections. The measurements, along with whatever cell morphology is visible, are enough to let me draw boundaries on my actual IHC sections. It may not be as perfect as it would be if I were to use a color channel for Nissl on my actual IHC-sections, but as long as the sections are from the same animals I feel like it’s close enough. ---
**Author:** ![achamess](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/achamess/32/534_2.png) [@achamess](https://forum.painresearcher.net/u/achamess)\ **Post date:** [October 31, 2017, 6:38pm UTC](https://forum.painresearcher.net/t/how-do-people-accurately-and-methodically-draw-laminar-boundaries-in-spinal-cord-ihc-images/318/3 "2017-10-31T18:38:48Z")
That’s great Liz. Thanks! What features are you using with the Nissl to call each lamina? Density? morphology? Would you be willing to post an annotated example from one of your own? ---
**Author:** ![liz](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/liz/32/267_2.png) [@liz](https://forum.painresearcher.net/u/liz)\ **Post date:** [October 31, 2017, 7:03pm UTC](https://forum.painresearcher.net/t/how-do-people-accurately-and-methodically-draw-laminar-boundaries-in-spinal-cord-ihc-images/318/4 "2017-10-31T19:03:02Z")
Our work is mainly focused on development or young ages of animals, so we primarily referred to this paper (Fig. 4 is particularly helpful): [http://onlinelibrary.wiley.com/doi/10.1002/cne.21691/full](http://onlinelibrary.wiley.com/doi/10.1002/cne.21691/full) This paper examines rat development, but the morphological traits hold up pretty well to mice also. I use a combination of cell size, morphology, and density/distribution to determine the boundaries of the laminae. I can show an example, from a P21 mouse, where we were concerned with lamina I-III, including the distinction between IIo/IIi. Therefore, there are 4 layers annotated on the Nissl image corresponding to each of these regions. ![1 20X Nissl](https://canada1.discourse-cdn.com/flex030/uploads/painresearcher/original/1X/20f4b73233fd411a7265d86e7535402425558802.jpg) ---
**Author:** ![achamess](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/achamess/32/534_2.png) [@achamess](https://forum.painresearcher.net/u/achamess)\ **Post date:** [October 31, 2017, 7:06pm UTC](https://forum.painresearcher.net/t/how-do-people-accurately-and-methodically-draw-laminar-boundaries-in-spinal-cord-ihc-images/318/5 "2017-10-31T19:06:57Z")
That’s extremely helpful Thanks Liz! I use fluorescent Nissl stain, but not all the time: > [@Stuff I tried during grad school/post-doc that won't get published but could be useful](http://forum.painresearcher.net/t/stuff-i-tried-during-grad-school-post-doc-that-wont-get-published-but-could-be-useful/302/14): > > For pan-neuronal labeling in the DRG, I’ve moved to Neurotrace, which is a fluorescent Nissl stain. The different colors have variable staining qualities. Here is Neurotrace Blue. I used 1:200 diluted in PBS for 10 mins at room temp. I do a quick wash in PBS and then mount. I’ve found the Far red Neurotrace to be good too. The Green Neutrotrace is very strong, but it also stains non-neuronal nuclei too (like real Nissl). I’ve found the blue and Far red to do th… I’ll start doing it again. Thanks for the annotation. ---
**Author:** ![tberta](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/tberta/32/145_2.png) [@tberta](https://forum.painresearcher.net/u/tberta)\ **Post date:** [October 31, 2017, 9:16pm UTC](https://forum.painresearcher.net/t/how-do-people-accurately-and-methodically-draw-laminar-boundaries-in-spinal-cord-ihc-images/318/6 "2017-10-31T21:16:08Z")
I use an approach similar to Liz, but I generally combine Nissl and IB4 fluorescent staining to determine the different laminae (see picture below). Lamina I is just 1 neuron or 2 small neurons at the upper border of the gray matter, then lamina IIo is down to the IB4 staining and IB4 staining is lamina IIi. Lamina III is usually the same size of lamina II (o+i). This staining can be performed with free floating SC sections in just 30 min using the IB4-Alexa 647 (1/200, Thermo, # I32450) and Nissl-Alexa 500 (1/400, Thermo, # N21480). ![SC_laminae](https://canada1.discourse-cdn.com/flex030/uploads/painresearcher/original/1X/9c1dd9435cbfa8cdc9d8f195aed70322f2695a52.jpg) ---
**Author:** ![achamess](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/achamess/32/534_2.png) [@achamess](https://forum.painresearcher.net/u/achamess)\ **Post date:** [October 31, 2017, 9:41pm UTC](https://forum.painresearcher.net/t/how-do-people-accurately-and-methodically-draw-laminar-boundaries-in-spinal-cord-ihc-images/318/7 "2017-10-31T21:41:17Z")
Thanks @tberta. Also very useful. When you say: > then lamina IIo is down to the IB4 staining and IB4 staining is lamina IIi. Do you mean that the cells between lamina I (1 cell thick) and the topmost margin of the IB4 staining is lamina IIo? And then the neurons that are within the purple band of IB4 staining, that’s lamina II inner? Nice staining! ---
**Author:** ![tberta](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/tberta/32/145_2.png) [@tberta](https://forum.painresearcher.net/u/tberta)\ **Post date:** [October 31, 2017, 9:49pm UTC](https://forum.painresearcher.net/t/how-do-people-accurately-and-methodically-draw-laminar-boundaries-in-spinal-cord-ihc-images/318/8 "2017-10-31T21:49:38Z")
Correct - the IB4 purple band is lamina IIi. ---
**Author:** ![zhzhj131421](https://avatars.discourse-cdn.com/v4/letter/z/f6c823/32.png) [@zhzhj131421](https://forum.painresearcher.net/u/zhzhj131421)\ **Post date:** [November 7, 2017, 2:34am UTC](https://forum.painresearcher.net/t/how-do-people-accurately-and-methodically-draw-laminar-boundaries-in-spinal-cord-ihc-images/318/9 "2017-11-07T02:34:08Z")
Hi, I think It is according to the cell size and intensity to bound the laminae > > achamess > > October 31 | Many papers that do spinal IHC in the dorsal horn superimpose boundaries on the laminae. ([Sensory Processing in the Deep Spinal Dorsal Horn of Neurokinin-1 Receptor Knockout Mice | Anesthesiology | American Society of Anesthesiologists](http://anesthesiology.pubs.asahq.org/article.aspx?articleid=1944809)) How do people consistently and accurately do this? What are the features or landmarks that you use? I know that certain molecular markers define laminar areas, for example PKC-gamma and lamina II inner and IB4 as lamina II outer. But in the absence of these markers, how do people do it? Neuronal density? that means you need to always use NeuN or Nissl. But that then takes up a color channel. What do you do? And what methods are there in the literature? @cedric.peirs@tberta@zhzhj131421@esypek@liz@janehartung Visit Topic or reply to this email to respond. To unsubscribe from these emails, click here.