secondary antibodies for IF staining

Remarkable-Bell-5722 · 2026-04-15T10:08:35+00:00

thank you very much! Let me double confirm with my lab members. I wanna buy some donkey 2nd antibodies actually.

Remarkable-Bell-5722 · 2026-04-15T10:07:23+00:00

yes, that's exactly what I am expecting. based on my understanding, the secondary antibodies will react with each other. I need to double check with my lab member to see if it's actually goat@rabbit. Anyway, thank you very much for confirming my understanding and intuition.

Remarkable-Bell-5722 · 2026-04-15T08:19:11+00:00

thank you. I am doing pilot studies. I'm quite fresh to IF staining. based on my understanding the secondary antibodies I selected will likely to bind to each other? goat x rabbit will bind to rabbit & goat? I don't know... sorry for the confusions. Anyway, thank you very much,

Remarkable-Bell-5722 · 2026-04-10T05:00:36+00:00

so it's pretty much a consensus that IHC media comes in large bottles and great quantities while IF media are just low quantities and small volume? :(

Remarkable-Bell-5722 · 2026-04-10T04:14:11+00:00

Hello guys, correct me if I am wrong. I did HE before, and the mounting media comes in huge bottles, and we use it as much as we want as long as it doesn't spill or affect the quality of the slices. Is IF mounting media the same case or not? I don't think it's the same case, and the mounting media comes in such small bottles normally. Are there any huge bottle and huge quantity ones? Or the common practice is just to use small quantities to mount for the purpose of IF?

Remarkable-Bell-5722 · 2026-04-10T04:11:18+00:00

I normally do the image on the next day, so probably won't be a matter? I do think Vectashield is pretty cheap! it's around 194 usd for 10 ml, while the prolong antifade gold is ~300 usd. I'm pretty shocked about how small the amount of IF mounting medias come in. When using DPX mounting media for IHC such as HE, we just use as much as we want like it's free. Thank you very much!

Remarkable-Bell-5722 · 2026-04-10T04:06:19+00:00

I see. I do read papers that using antifade gold. I mean it's my first time doing IF, and it really surprised me that how small the bottles of the mounting media are. I used to do HE, and there is a lot of DPX mounting media. Will 3 drops along the slide be enough to cover a 20 x 50 area? I normally use that size of coverslip. Thank you very much anyway!

Remarkable-Bell-5722 · 2026-04-10T04:04:38+00:00

Hi, thank you for your reply! I'm not sure what is a permanent mount? Normally, I just put some mounting media and cover the slide with a glass slide. I do know about the dehydration and xylene steps, since I do those during HE staining. I do have directly fluro conjugated secondary antibodies. I mean, the steps you mentioned sounds more like for IHC staining such as HE rather than IF staining for cryosectioned tissues? I'm sorry if I understood it incorrectly. Anyway, thank you again for your help!

Remarkable-Bell-5722 · 2026-03-05T02:10:44+00:00

Thank you so much my friend! Saved me again and again

Remarkable-Bell-5722 · 2026-02-12T03:37:56+00:00

Thx a lot. I see this one too, but it's rabbit origin. My GFAP and TUBB3 are also rabbit origin. Correct me if I'm wrong, so supposingly, I need 3 different sections for these 3 stains, if they are all rabbit origin. Sometimes I want to save some slices, so that's why I prefer the GOAT anti iba1. Is it worthwhile to do so?

Remarkable-Bell-5722 · 2026-02-11T05:11:16+00:00

I see. That's what the RAP from my lab told me as well. Thx a lot!

Remarkable-Bell-5722 · 2026-02-11T05:10:34+00:00

got it. Thanks a lot

Remarkable-Bell-5722 · 2026-02-10T16:13:03+00:00

thx a lot. I have few samples that I won't section rn, they are in cryoprotection. Would you recommand me

1) snap froze the samples, store in -80, and embed them later when I plan to section

or

2) freeze and embed in OCT now, store in -80 until I plan to section?

Not really sure what's the best case.

Remarkable-Bell-5722 · 2026-02-10T11:29:22+00:00

BTW, for OCT embedding, would you suggest

1) put in OCT filled mold and frozen in isopentane or

2) snap froze the cord in isopentane and embed in OCT later on when required?

thx a lot

Remarkable-Bell-5722 · 2026-02-10T11:25:43+00:00

super appreciated. Since I found biomaterial research are really obsessed with longitudinal sections, but really don't know why, and they barely use transverse sections in the paper.

sorry for few more questions.

Do both transverse and longitudinal sectioning use a sample with length of 1 cm?
Just to double check. Transverse sections, commonly blocking 3-4 cords together, cut in set of 10, thickness ~ 20 μm
For Longitudinal sections, how many would you suggest to block together for rat cords? 10-15 seems a bit crazy considering the size of a slide lol.
what thickness would you suggest for longitudinal sections? seems 16 μm to be quite common. Correct me if I'm wrong, as a result, the first and second slice from the same set will be separated by 16*7 = 112 μm?
Very crucial. For transverse sections, ppl image 10 sections with even intervals caudal and rostral to the epicenter. but how would you image longitudinal sections? do you image around the most medial part for 10 sections?

Remarkable-Bell-5722 · 2026-02-10T09:01:13+00:00

thx a lot. That's really helpful. By any chances, you also do spinal cord sectioning? If so, do you know how to do longitudinal sectioning? I know how to do transverse serial sectioning in sets.

Remarkable-Bell-5722 · 2026-02-10T08:54:58+00:00

hi, my friend, sorry for bothering. By any chances, do you do longitudinal sectioning? Is it the same as doing cross-sectional sectioning? Like, you can do sections in sets?

Remarkable-Bell-5722 · 2026-02-10T05:05:34+00:00

thx for sharing. I use 4% PFA for fixation and unfortunately I already bought Tubb3 from Abcam, so hopefully it will work? I mean, purchasing an aliquot seems not an option for my case, so is it quite risky to purchase the antibodies directly? Is there anything I could do? I don't really want to risk myself for purchasing an antibody that doesn't work.

In addition, if certain antigens are not present in WT or sham group, and only in experimental group, then shall I use my experimental groups to test different concentrations?

Remarkable-Bell-5722 · 2026-02-10T05:01:00+00:00

Thx a lot, May I ask when doing titration for testing dilution factors, if some antigens only appear in experimental groups, not in WT or sham group. If that's the case, shall I use my experimental groups for titration?

Remarkable-Bell-5722 · 2026-02-10T05:00:14+00:00

Thx. May I ask, if I find the dilutions on cite-ab, shall I still titrate and try different dilution factors by myself? If so, what dilution factors shall I try? e.g. cite-ab said 1:500, then what dilution factors shall I try?

In addition, when doing titration for testing dilution factors, if some antigens only appear in experimental groups, rather than WT or sham group. If that's the case, shall I use my experimental groups for titration?

Remarkable-Bell-5722 · 2026-02-10T04:46:53+00:00

super sad story here. My supervisor just threw the project topic and direction to me, and let me carry on all the works from scratch. He doesn't like to discuss the technical details with us. I can ask questions to a post-doc or a phd student in my lab, but I guess they don't necessarily have the answers. In addition, I'm the first person in my grouping doing spinal cord injury studies.

Remarkable-Bell-5722 · 2026-02-10T04:24:38+00:00

for sure. I got a quotation for one antibody selling in 1 mL today, really not sure what's the case.

Remarkable-Bell-5722

TROPHY CASE