Talk:Immunolabeling

message: This article only says that the secondary antibody can be tagged, but the article Direct fluorescent antibody indicates that the primary antibody can sometimes be tagged. M-1 (talk) 11:03, 14 April 2012 (UTC)
replies: text: Correct. Secondary antibody tagging has the major advantage of cost, the way it's usually done. One can produce a "normal" (untagged; primary) antibody for the specific antigen of interest, then use a mass-produced tagged secondary antibody to locate it. Otherwise, one would have to not only get the initial primary antibody, but do the work to get it properly tagged for localization, which would likely cost more and take more time. I'll try to locate some references on this that are accessible to others... Allens (talk | contribs) 22:14, 27 May 2012 (UTC)
thread_title:  Primary antibody tagging 

message: Greetings, fellow Wikipedians!  This article needs to be greatly improved in the following ways:
:* Add verified references and inline citations within the lead section 
:* Add free copyright images throughout the article for a better visual understanding of this technique
:* Provide some general statements (with references) within the lead section for any reader to have a better grasp of its importance
:* Provide subsections after the lead section
:* Overview of antibodies and their role in immunolabeling
:* Provide examples where immunolabeling is used
:* References, references, and more references
:* If feasible, find a free copyright video of the technique being used
Jbmontgomery24 (talk) 00:27, 13 March 2013 (UTC)
thread_title:  Improvements in Need for the Immunolabeling Article 

message: Since this article is being created within the context of already existent articles on immunocytochemistry and immunohistochemistry, I recommend that the focus of this article should be on the purposes of immunolabeling in general and the fundamental tools: Ab, tag, signal resolution, etc.
I recommend avoiding any detailed discussion of the procedures that would overlap with the articles mentioned.
My initial thoughts on the structure of the lead are as follows: 
   
1.) I like the first sentence. I suggest the following sentence indentify, distinguish and link to the immunocytochemistry and immunohistochemistry subsets. This will signal to the reader that immunolabeling is a very broad procedure; the essence of which involves just a few elements.
2.) Explain the type of information that can be obtained through immunolabeling.
3.) Identify the elements and distinguish between the direct and indirect approach.
4.) Explain why the indirect approach is more practical.
5.) Briefly describe the binding dynamics of antibody and how they are produced.
6.) Briefly describe some common tags and how they are fused to secondary antibody.
7.) Discuss the resolution of the tag signal.Jakodak (talk) 13:08, 13 March 2013 (UTC)
thread_title:  Suggestions for Lead Section Structure 

message: Hello all!  I currently have the opportunity to help with this article's status through a Molecular Biology course that I am taking through a graduate program at JHU.  For this project, my partner (Jakodak) and I will do our best to continue to develop this article.  A lot of editing will be necessary in the lead section due to the lack of references.  Anyone oppose to the lead section being redone immensely due to no sources in this section? If not, we will start our new additions and edits shortly.  Thanks! Jbmontgomery24 (talk) 22:38, 31 March 2013 (UTC)
thread_title:  Lead Section Edits 

message: I think that overall your article was very informative. I definitely learnt a lot about "immunolabeling"! It's very cool that you were able to get permission from Dr. Thomas Caceci to use his images using the "immunolabeling" technique. I can't wait to see them.
Following are a few small edits:
* Issues with singular and plurals: use "piped links" to solve problem.
Antigens are organic molecules, proteins, capable of binding to antibodies. Linking antigen-specific antibodies with a means of detection, known as a tag, provides a way to observe the location of the antigens to which the antibodies bind. If the immunolabeling process is meant to reveal information about a cell or its substructures, the process is called immunocytochemistry. Immunolabeling of larger structures, such as biological tissues, is called immunohistochemistry.
There are two complex steps in the manufacture of antibodies for immunolabeling. The first is the production of antibodies that bind specifically to the antigen of interest and the second is the fusion of the tag to the said antibodies. Since it is impractical to fuse a tag to every conceivable antigen-specific antibody, most immunolabeling processes use an indirect method of detection. This indirect method employs a primary antibody that is antigen-specific and a secondary antibody fused to a tag that specifically binds the primary antibody. This indirect approach permits mass production of secondary antibodies that can be bought off the shelf.[1]
*3rd paragraph: Add enzyme link
*4th paragraph: question to myself: if the gold is so dense that it is impenetrable, why is there radiation from it? Wouldn't it be more of a black spot?
*Very good 5th paragraph!
*8th paragraph: extra space at end of: "... haptenylated primary antibodies can be used ,"
*9th paragraph: missing some links for "tag" & "antibodies"
*10th paragraph: For references, should there be a space before the number or not? (I'm not sure.)
MartinLubell (talk) 06:17, 11 April 2013 (UTC)
replies: text: Martin, thank you for all your editorial advice.  I went ahead and corrected the spacing in the 8th paragraph.  Good eye!  I think you are right on the spacing before the number of the references.  Those have been corrected.  My partner and I will definitely consider your other suggestions as we continue to work on this article over the next few weeks. Thanks, again! Jbmontgomery24 (talk) 12:22, 11 April 2013 (UTC)
replies: text:  You are very welcome! Ciao, MartinLubell (talk) 12:37, 11 April 2013 (UTC)
replies: text: Martin, thank you for your thorough review of our article, for we sincerely appreciate your time.  All of the bulletins that you have mentioned above have been edited/addressed by us except for your comment about paragraph 4.  Since my partner focused on that area of the article, I'm sure Jakodak would be more than willing to give you more insights about your questions during our continual discussion.  In addition, since Dr. Thomas Caceci is his contact, he will most likely be adding the images to the article very shortly, as it will add more visual interpretation of the technique.  We hope you have found the article an efficient learning tool thus far, and we will continue to improve it.  Kind regards! Jbmontgomery24 (talk) 18:28, 24 April 2013 (UTC)
replies: text: I think your article is VERY solid. It goes over all aspects of its domain. Unless I'm missing something, I think it will get the highest grade! MartinLubell (talk) 15:50, 25 April 2013 (UTC)
replies: text: Thank you for the kind words, Martin!  This article could have not been completed without the efforts of our group collaboration, the OAs, our professor, and the assistance of the reviewers.  I'm sure there will be more edits and tweaks as we proceed with the project, but hopefully, we have a concrete foundation on the topic.  Again, if you see anything else, please feel free to jump in!  Thanks! Jbmontgomery24 (talk) 16:11, 25 April 2013 (UTC)
replies: text: Martin, this is just an FYI - according to our OA (Keilana), we don't need a space between the reference and the punctuation at the end of the sentence.  Thanks! Jbmontgomery24 (talk) 17:41, 7 May 2013 (UTC)
replies: text: Hello Jessica, are the images at the bottom the ones from Caceci? (They don't have citations.) If they are, you could try to place at least one of them in the article directly. Have a great summer, and till next class together! MartinLubell (talk) 19:23, 9 May 2013 (UTC)
replies: text: Hi, Martin!  No, the images at the bottom were part of the article when we started on the stub.  The only item that we added at the bottom was "Immunolabeling as a tool for understanding the spatial distribution of fiber wall components and their biosynthetic enzymes" per David's finding during his review.  My partner added the image in the lead section; however, he created that using Dia software.  Since the deadline isn't until tonight, my partner may still be adding images to the article from his contact, Dr. Caceci.  I agree - if there are images added, they will have citations associated with them.  Thanks for checking in.  Best of luck to you as well!  I'm sure I will see you in another course.  Cheers! Jbmontgomery24 (talk) 21:13, 9 May 2013 (UTC)
thread_title: Comments from MartinLubell about Immunolabeling Wikipedia article

message: Immunolabeling
* Keep in mind that my comments are just suggestions -- don't feel compelled to take my advice if you disagree with it.
* The lead is a too long.  Check WP:Lead for guidance.  You don't need all the details that are in the 2nd, 3rd, and 4th paragraphs -- they should be moved into the main article body.  Also, in the 5th paragraph, just a summary line or two about the applications is enough to establish why these are notable; the details can be moved into the article body.
* Don't link the same word twice in a very short space, as you did with antibody.  Check WP:Link for an explanation of when and when not to link.
* In section headings, only capitalize the first word, not every word.
* Don't use bold for emphasis.  Use italics (like this: italic text) as described in Wikipedia:Manual_of_Style/Text_formatting#Emphasis.
* Under "Antibody Binding and Specificity", way too many links.  Again, check WP:Link for some guidance.  In general, don't link the same thing more than once (except, sometimes it's okay to link it once in the lead and once in the body.)
* It looks to me like "Immunolabeling for Transmission Electron Microscopy" and "Immunolabeling for Scanning Electron Microscopy" should be subsections of "Immunolabeling for Electron Microscopy", not siblings.
* Under "Specific immunolabeling techniques", I am confused, because you only have sections for electron microscopy, yet you mention light microscopy.  Shouldn't there be a section for that?
* More figures could do a lot to help elucidate this topic.
Klortho (talk) 03:21, 26 April 2013 (UTC)
replies: text: Hi, Klortho!  Bulletins 3-8 have been addressed.  Hopefully, the article demonstrates your suggestions well.  My partner focused on the lead section and has images to add to the page, so once he addresses those issues, the article will show more improvements.  Please feel free to let us know if there is anything else that needs to be added and/or edited.  Thanks very much for your suggestions! Jbmontgomery24 (talk) 15:19, 26 April 2013 (UTC)
replies: text:  Hello Klortho, Thanks for reminding me of the link rule: once you have a word linked, you don't have to repeat the link. I had incorrectly suggested that, also for another user. MartinLubell (talk) 15:57, 3 May 2013 (UTC)
replies: text:  Looks much better! Klortho (talk) 18:34, 5 May 2013 (UTC)
replies: text: Thanks again for all your helpful suggestions along the way!  We greatly appreciate it! Jbmontgomery24 (talk) 23:52, 5 May 2013 (UTC)
replies: text: Klortho, I just had a quick question for you.  Since words are not supposed to be bold within the article, can the first word (Immunolabeling) of the lead section in the article be the only word in bold text?  Thanks for the help! Jbmontgomery24 (talk) 13:34, 6 May 2013 (UTC)
replies: text: Never mind.  I found this this was OK to do from another OA.  Thanks! Jbmontgomery24 (talk) 00:41, 8 May 2013 (UTC)
thread_title:  Comments from Klortho 

message: Hi, below are some thoughts after reading the Immunolabeling article. Overall, it is well-written, and kudos for tackling such a technical subject matter and making a good start in introducing it to the layperson. 
Strengths:
# Neutral point of view
# Well-organized; structure seems solid
# References are good, with a couple of minor suggestions noted below. After reviewing each reference, each seems to support the article content with no concerns
Areas for Improvement:
# The biggest areas in general would seem to be:
## Recommend cutting the length of the introduction. Consider cutting it down by simply summarizing each paragraph in this section in 1-2 sentences, saving the details for the main body. --This is one of my partner's focal points. I'm sure he would be able to address this. 
## There's a lot of scientific jargon throughout (specifics noted below) that would make it difficult for the layperson to absorb and follow. We may consider that the target audience is one who stumbles on the subject after reading the term "immunolabeling" in a major news story --My partner will be able to help out with this suggestion.
## The areas that came to mind that could be potential subjects to add include background on immunolabeling itself, such as how was it pioneered? By whom?--Once again, my partner would be best to address this.  However, I can put my two senses in that the history of immunolabeling was rather difficult to find in the literature since it is a technique that seems to be derived from multiple areas.  Therefore, it is hard to pin point the exact person or persons that developed this technique. 
# Specific areas:
## Indirect vs direct method: 
### Direct method needs an explanation --Additional information/reference was provided for this explanation.
### Can cross-reaction be described? --This was linked to cross-reactivity to give the reader a better idea behind this concept.
### "Covalently labeled" may need to be defined/explained Additional information/reference was provided on this term. 
### Last sentence of the 1st paragraph: "it can result in less sensitivity over direct method"; Do you mean indirect method here? It seems that this part highlights the advantages of the indirect method over the direct method.'--Good eye!  Yes, that is exactly what we meant.  It was changed it, so it states that the direct method is less sensitive than the indirect method.  Thanks!
### 2nd paragraph: there's a lot of scientific jargon that may be pared down a bit, or at least given more background and/or links provided. Examples are two-step protocols, secondary Fab antibodies, haptenylated primary antibodies, succinylimidesters, conjugated IgG Fc-specific Fab--This was difficult to do.  No more explanation was stated in the reference I listed, and these words cannot be linked to any words from Wikipedia.  I will see if my partner as better luck on this one.  I agree, though.  More explanation here will help the reader. 
## Antibody binding and specificity
### 1st paragraph: refers to items that may require more background, especially for the layperson being introduced to this material for the first time. Examples include: affinity column, epitope --These words were linked appropriately.
### 2nd paragraph: background may be needed for PTM, denatured, and immunoblotting --These words were linked appropriately. 
### 3rd paragraph: not clear how this relates to this section on binding and specificity; refers to items that may require background or explanation, such as HeLa cells, transfected, fibrillarin, green fluorescent tag; GFP refers to "green fluorescent protein" vs "green fluorescent tag" --Additional information was stated in the first sentence of this paragraph to explain the reasoning for having this paragraph in this section.  The concentration of an antigen can clearly affect the binding to its specific antibodies.
## Immunolabeling for light microscopy
### potential jargon word in "objective" --This word was linked correctly to assist the reader.
### 2nd paragraph: lot of jargon here; examples are histology inspection, picric acid-formaldehyde, human GH, PRL, TEM, SEM, immunogold --This was difficult to do, so this was part of the experiment that was conducted in the reference I found. Only some of the words could be linked.  TEM, SEM, and immunogold are linked later on in that section.  The other 3 words you mentioned above could not be linked.
### Please check the grammar of the sentence that begins "Moreover, 319 monolayer pituitary adenomas..." --Thank you.  This has been corrected.
### "adenoma" misspelled at the end of the paragraph This has been changed.  Good catch!
## Immunolabeling for electron microscopy
### 1st paragraph: not sure the 2nd sentence, which provides a brief reference to the fact 2 types of electron microscopes were invented about the same time, is necessary --This has been edited.  Thanks for noticing this. 
### 2nd paragraph: jargon in "wax block section" --This was completely taken out for simplicity purposes.
### 3rd paragraph: jargon needing explanation or links: chromosomes, topoisomerase Ila, condensin, mitotic --All linked properly.
## References
### Ref 10: links to the talk page of a wikipedia article; PMID link works, though --It is no longer reference 10 anymore since more references have been added.  It is currently refernce 16 (Fazekas, et al. (2005)).  However, the journal title is no longer linked, so the PMID is only linked.
### Ref 18: no link provided --This has been corrected.  It is currently reference 25 (Narayanan et al. (2011)).  
### Potential new reference: this seems to put the importance of immunolabeling in perspective (http://www.tappi.org/content/pdf/events/06NANO-papers/Session%203%20-%20Haigler.pdf)--What a great find!  Thanks so much!  This has been added to the external links since it has a nice overview of the techniques as well as all the visuals.
Of course, these are just suggestions. Please feel free to make any you deem appropriate, and don't hesitate to ask if you have any questions. Best, Davidwhanks (talk) 03:45, 2 May 2013 (UTC)
replies: text: David, thank you very much for your very thorough review on our article.  Most of your suggestions have been addressed, so please see the notes indicated after each bulletin.  There are only a few that still need to be addressed, but hopefully, the currents edits have polished the article a bit.  Please let us know if your eye catches any more edits or more suggestions arise. Thanks again! Jbmontgomery24 (talk) 17:06, 4 May 2013 (UTC)

text: David, thanks for the many helpful suggestions.  In addition to the excessive length of the introduction, a full and slow reading reveals numerous vague and unhelpful sentences.  Indeed, your exhaustive list seems almost too generous.  In response, a lot of the redundancy has been removed and many of the sentences rewritten.Jakodak (talk) 03:03, 9 May 2013 (UTC)
replies: text: Hi Jbmontgomery24 and Jakodak, thanks so much for the update. Your article looks really good! Congratulations! Cheers, Davidwhanks (talk) 03:23, 9 May 2013 (UTC)
replies: text: Again, thanks for your extensive review to better our article, and we're glad you approve of the improvements! Jbmontgomery24 (talk) 11:20, 9 May 2013 (UTC)
thread_title:  Comments from Davidwhanks 

message: Looking back through the comments, edits and changes over this course suggest an article that has been refined nicely.  Frankly, I don't have much more to say beyond what others have.  I have a few comments below which I believe will help people better understand the context, and comparative aspects of the techniques described in your article.  I've provided comments by section, hoping that will make review less complex.
Main section
*The phrase "usually a protein" should be "usually proteins" --This has been corrected.  Thanks for the catch!  
*It would be interesting to incorporate laboratory tests which use immunolabeling, like ELISA tests, quick strep/HcG tests, etc.  These are used in thousands of labs across the world. --This area was one of my partner's focal points, so I will see what his insights are on this bulletin.  
*IN intro, it would be helpful to have other examples (last sentence) and possible references in the details Three references were provided to support the different areas mentioned that use immunolabeling.  Please note the references are placed in the order that the fields of science were listed in the sentence. 
Indirect vs. Direct
*What are some of the problems with direct over indirect?  This statement is made, but there are no concrete examples comparing or contrasting the two methods. --This question has been addressed by the large expansion in this section.
*It would be helpful for the interested reader to have a pro/con type comparison between indirect and direct.  Perhaps a table or graphic? --Additional statements have been made to address the differences between the two methods; therefore, a table wasn't provided.  The literature focuses more on the pros of using the indirect methods, so it was difficult to find the pros of the direct method.
Antibody binding and specificity
*Suggest linking monoclonal and polyclonal.  I would think a curious reader may want to know what these are. --Nice suggestion - this have been addressed.
*The second paragraph wanders.  Suggest rephrasing content around antibody specificity and the need for purification? --The paragraph was edited more towards the beginning to give the reader a better overview of how essential the synthetic peptides and purified proteins are in the specificity of antibodies.  The paragraph is as simple as it could be based off of the reference, meaning the reference explained this in a much more complex manner.  The best efforts were put forth for this issue.
Immunolabeling sections
*How sensitive is light vs. electron, etc. microscopy and immunolabeling?  How do these compare?  It might be nice to provide some type of comparative aspect to this section --An additional sentence and reference was added to the EM section to explain the difference in their magnification (EM is much more sensitive than light microscopy).
Enjoyed reading this article, and look forward to seeing it continue to improve.  Thanks! Aaron.aude (talk) 02:02, 3 May 2013 (UTC)
replies: text: Aaron, thank you very much for your suggestions, for we greatly appreciate them to improve our article.  Please see the notes after each bulletin.  Almost all of your bulletins have been addressed.  The only one remaining is the second bulletin for the lead section, but that was one of the areas that my partner focused on.  Perhaps, he can give you more insights on your comment?  If you see any other minor error or have suggestions, please feel free to let us know.  Thanks again for your thorough, helpful review! Jbmontgomery24 (talk) 16:32, 4 May 2013 (UTC)
thread_title:  Comments from aaron.aude 

message: Hey guys! Here's my review - ask me if you have any questions about anything I've suggested to you.
* The lead is really long for an article of this size. Try to put some of the information in the lead in the body of the article. --My partner will be making these edits soon since it is one of his focal points.
* Also you don't need any footnotes/citations in the lead if the information in it is covered in the rest of the article (which, in most cases, it should be).  --Some of the citations will remain within the lead that are not part of the main body of the article.  However, my partner will be rearranging parts of the lead; therefore, some of the citations may move accordingly.
* The first image could do with a more explanatory caption. --This has been corrected.
* Check throughout for this - references should come after punctuation and there should not be a space between the punctuation and the reference. --This has been corrected.
* I don't see source 2 (Matos et al.) saying that immunohistochemistry is for larger molecules; they say that it can be used for amino acids. Am I just missing something? --Within, the abstract, it states that it can be for smaller molecules (amino acids) to larger molecules (proteins).  The sentence structure was tweaked here to incorporate small molecules as well.
* This sentence needs some tweaking: "The first is the production of antibody that binds specifically to the antigen of interest and the second is the fusion of a tag to said antibody." I think it should either be "of an antibody" or "of antibodies that bind" for subject/verb agreement. --Good eye!  It was changed to "of an antibody". 
* Not all of the information in those two lead paragraphs is on the specific page (1066) of source 3 (Oliver); did some of that come from elsewhere in the book? If so, cite those pages too. --My partner will be able to assist with this area.
* Can you explain how epitopes are effective tags? All of the others are obvious in why they're visible. --A brief explanation was added as well as a reference to this section.
* You use a ton of parenthetical phrases throughout, you should try to rewrite some of those. --Many of the parentheses were removed, only some remained where it was appropriate.
* If you could have more than one source in the "Indirect vs. direct methods" section, that would be fantastic. --There are four sources in this section.
* Italicizing words is unnecessary here. You could link a couple of those terms though. --This has been corrected.
* Succinylimidesters and conjugated IgG Fc-specific Fab sections need to be glossed. --Many of those words were linked to give a better understanding.  An outside dictionary source was used to define succinyl.  The true definition of succinylimidesters was hard to come by.
* The last paragraph of "Antibody binding and specificity" needs a spell check and copyedit. --It has been spell checked and copyedited.
* Don't capitalize both words of "Light Microscopy". --This has been corrected.
* The sentence beginning "Moreover, 319 monolayer cell cultures" contains language directly taken from the paper it is cited to. Please rewrite, preferably with more accessible language. --This has been edited as well as simplified for the general reader.
* The first paragraph of "Immunolabeling for transmission electron microscopy" has one sentence that is too closely paraphrased from the book and one that is directly taken from that book. Please rewrite. --This has been corrected.
* The sentence beginning "A study was conducted to view the morphology" is far too closely paraphrased; this occurred throughout the paragraph. Please rewrite. --This has been edited. 
* The link to the paper "An immunocolloid method for the electron microscope" does not work. PMID ID has been left since the link does work.  There just isn't a summary for the abstract here.  However, a doi link was added as well, but there isn't much of a description on this site as well.  My guess is since this paper was written in 1971, there isn't much info on these sites, even though it is a fundamental paper.
:Please see the comments next to each bulletin listed above.  My partner will be able to close the gaps on some of the suggestions mentioned, so once he does that, the article should show more improvements.  If there are any more suggestions and/or questions about the comments listed next to each bulletin, please let us know.  Thanks for all your great insights! Jbmontgomery24 (talk) 17:34, 7 May 2013 (UTC)
replies: text: Keilana, the Matos et al. reference was completely omitted from the article and replaced with a more sufficient reference pertaining to proteins (larger molecules) only.  Hopefully, this fits in that area of the article more appropriately.  Thanks! Jbmontgomery24 (talk) 18:47, 7 May 2013 (UTC)
replies: text: Keilana, in addition to the specific changes addressed by Jessica, I have made the fundamental rewrites required of the first three sections.  Hopefully, you are watching this page and will be able to see for yourself the improvements prior to the end of the course.  I believe the wording throughout the article is now much clearer and more able to effectively teach the reader.Jakodak (talk) 03:17, 9 May 2013 (UTC)
thread_title:  Review by Keilana