I recently performed H&E staining on my paraffin embedded P0 mice tissues to visualize the phenotype of the eye in one of my WT mice. I managed to slice the tissues and stained them with Hematoxylin & Eosin dye. However, I realized that there were cracks (on the retina in particular) on my sliced tissues (please see attachment to get a clear view of my problem)
I am aware that the cracks on the eye retinal may have arised due to over-dehydration of the tissues. However, I am not sure at which particular steps the over dehydration may have occurred? As such, I have briefly described the processes involved in my H&E staining for you to comment:
1) Collect tissues and wash them in chilled 1xPBS solution (x3)
2) Tissues were fixed with Bouin solution and incubated overnight at 4°C
3) Bouin solution was discarded and samples were washed thoroughly with 70% Methanol until solution becomes light/almost colorless
4) Dehydrate tissues with 80% Methanol overnight at 4°C
5) Dehydrate tissues with 90% Methanol overnight at 4°C
6) Dehydrate tissues with 100% Methanol at 4°C for 6 hours
7) Dehydrate tissues with 100% anhydrous Methanol at 4°C for 6 hours
8) Dehydrate tissues with 100% anhydrous Methanol overnight at 4°C
9) Displace tissues in Chloroform at RT for 2 hours (x3)
10) Displace tissues in paraffin at 68°C for 3 hours (x2)
11) Displace tissues in paraffin at 68°C for overnight
12) Embed tissues in paraffin
H&E Staining:
1) Wash samples with 1xPBS (5mins x3)
2) Soak tissues in Hematoxylin solution (1 minute)
3) Wash samples with running tap water (5 min)
4) Soak samples in 0.1% Eosin solution diluted in 80% Ethanol (1 min)
5) Dip samples in water and move up and down
6) Soak samples in 90% EtOH (1 min)
7) Soak samples in 95% EtOH (1 min x 2)
8) Soak samples in 100% EtOH (3 min x 2)
9) Soak samples in Xylene (5 min x 3)
10) Mounting
I have attached my H&E stained eye section for your attention and comments on how I can improve my technique. Also, please advise if the intensity of the staining is appropriate (i.e: hematoxylin vs Eosin).
Thank you
The first thing I would try
First where is the cracking occurring - at the pre- or post staining stage?
If pre- staining then most likely the water bath is too warm. When you spread the ribbon it is over spreading.
If it is post-staining, then it is likely the xylene. Try a clearing agent like cederwood oil (or a modern equivalent is histoclear) which hardens the tissue less.
Two or three additional imponderabilities:
first of all: why you use methanol as alcoholic dehydration agent? (is it because of its easier availability or because of a certain improbvement of the processing?.... Usually for dehydration in histological studies will be used either Isopropylalcohol=iso-Propanol or, -better- (denatured) ethanol, secondly, you dehydrate (steps 6-8) in 100% absolute [in my understanding this is "waterfree" ) methanol at 4 ° C. Finally then, as an intermediate you use(d) chloroform (normally xylene is used).
You are aware that 4°C ABSOLUTE 100% methanol is "absolutely" hygroscopic ?? and if changing to "absolutely water-immiscible" chloroform at RT (out from 4°C ) you'll for sure protract water traces in your specimens which may turn out as "problem zones" during embedding into paraffin and might be responsible for separation (cracks) of the retinal layer(s) in your specimens.
Would you mind to give a reference for the processing schedule you adhere to? I should be glad to find out, to learn about and to have documented that in my e-library (at least for "histological whole eye processing"), why methanol and chloroform is used in the process instead of common usage of Iso-Propanol or Ethanol and Xylene. Also, perhaps Davidson’s fixative*) see below /bottom - would be a better alternative for fixation compared to Bouin's.... Thank you, best wishes and good luck !
NB: read and find [CTRL-F and insert] the word | under-processing | or also | over-processing | in: LEICA-PATHOLOGY LEADERS brochure, "Difficult Blocks
and Reprocessing - What To Do When Your Block Won’t Section "https://www.leicabiosystems.com/fileadmin/biosystems/PDF/95.9890_Rev_C_Difficult_Blocks_and_Reprocessing.pdf
Davidson's fixative solution, need 2x 100 ml
2 ml formalin (37% formaldehyde)
10 ml glacial acetic acid (99.7%)
35 ml ethanol (98%)
53 ml distilled water
Place each eye in 100 ml of Davidson's fixative solution and leave to post-fix at room temperature.
After 18 - 36 hr in Davidson's fixative, transfer to (100ml) 50% ethanol (room temperature) for 6 - 8 hr, then finally to 70% ethanol (room temperature).
Refrigerate samples at 4 °C and store until dissection. (cf: https://www.jove.com/video/50411/techniques-for-processing-eyes-implanted-with-retinal-prosthesis-for )
cf. also: https://biospecimens.cancer.gov/meeting/brnsymposium/2011/Posters/Lituev-508.pdf
or read/cf: ARKO-BOHAM et al, 2014: Improved Method of Producing Satisfactory Sections of Whole Eyeball by Routine Histology, @ https://de.scribd.com/document/298717314/tmp8320-tmp .
Only or a comparison (:-)) a historical paper by BARRETT, 1886: http://jcs.biologists.org/content/joces/s2-26/104/607.full.pdf
Dear Dr. Jones,
Sorry for my late response and thank you very much for your comment to my post. After reading your comment last week, I instantly checked some of my unstained slides. I compared the slides before and post staining and observed that the cracks on the retina emerged only after performing staining (Images attached for your perusal). The protocol that I adhere to is a well established in house-lab protocol. Previous researchers in our laboratory had been following the similar protocol and they were able to get decent images without retinal cracks. Hence, I do not think that Xylene maybe causing the cracks?
Dear Dr. Wolfgang,
Thank you for your insightful comments. I adhere to our well established in house-lab optimized protocol, hence, I do not have any reference to give you. I am sorry. The Davidson's fixative method looks pretty promising as well. I am also parallely trying out this method.
Thank you
- Badges
- Science topic
- Similar topics
- Methodology
- Laboratory
- Histological Techniques