Order Description
-Introduction:
This exercise aims to involves the student in the procedure of preparing a tissue section from the beginning of dissecting the rat till the last step which is examine the tissue microscopically and detect whether the tissue that has been taken from a specific organ is normal or not.
1-Dissecting the rat and fixating the sections
Firstly, every three student shared one rat that has been given by the demonstrators. The first student was dissecting the rat to obtain the wanted organ and the second student was sectioning each organ and giving each student a section to put it in the cassettes that have been labelled with the name of each organ, student by the third student. Each student has obtained a section that fit appropriately into a labelled tissue cassette from each organ and the organs are Kidney, Liver, Lung, Duodenum, Oesophagus and trachea, then all the cassettes were fixed in a 10 % normal buffered formalin. The fixative step is an essential step and it must be applied immediately after collection to avoid osmatic swelling, purification, autolysis, shrinkage and loss of cellular compound of the tissue(1).
2- Processing:
The prepared cassettes were taken to be processed by the Leica ASP200S tissue processer. The concept of processing the tissue is to remove the water from the sectioned tissue and replace it by a medium material that solidifies the biological tissue to facilitates cutting thin sections. After fixing the tissues into a 10 % normal buffered formalin for at least 8 hours, the tissues were dehydrated to remove as much as possible of the water that in the tissues, so the tissues go through a series of a dehydration steps. The first one is putting the tissues into a 70% ethanol for 15 minutes then into a 90% ethanol for 15 minutes then into a 100% ethanol for 15 minutes than into another 100% ethanol for 30, at the end they are put into the last 100% ethanol for 45 minutes, after that the water should be removed from the specimen. Then the Clearing agent is applied to displace the ethanol in the tissues with molten paraffin wax, this step has another aspect rather than replacing the ethanol with paraffin wax which is removing the fat from the tissues to avoid presenting the fat as a barrier to wax infiltration. Moreover, this step is done by imbued the tissues in xylene for 20 minutes, then moving them to another xylene for 20 minutes, eventually moving the tissues to another xylene for 45 minutes. Finally, the tissues are infiltrated with an optimal wax that melted under 60 °C. This is done by Embedding the tissues the wax for 30 minutes, then moving them to another wax for 30 minutes, then moving them to another one for 45 minutes. In addition, removing mineral from bone or other calcified tissue” Decalcifications “was done to the femur section after to enhance cutting bone sections by the Rotary microtome (1).
3- Embedding:
This step was done by utilizing a mould paraffin wax for preparing the tissue to be cut by the Rotary Microtome. After the tissues have been fixed, they were taken to the Heated Paraffin Embedding Station to prepare the tissue to be settled at the right position in a wax block. HistoCore Arcadia H&C was used to do that by taking off the tissue from the cassette to a suitable mould and fill it with a little amount of the wax, then place the tissue into the right way so the cut surf faces down and move the mould to the cold spot to assess tissue orientation, then the mould was covered by the cassette, filled up with melted wax and moved to the cooling plate of the Arcadia C. The cool plate is held at a constant temperature of -6 degrees (4).
4- Cutting:
Afterwards the prepared wax blocks are taken to be cut by the Rotary Microtome. The Rotary Microtome must be cleaned up and checked whether the knife is good or not and all the cutting equipment are available. Furthermore, the water bath must be settled at 37 degrees and clean. All the blocks were put on a cold surface such as an ice block to prevent shrinking the ribbon when they are cut out and transfer to the water bath. An important step must be applied once cutting process is starting which is trimming. To trim the block, the Microtome must be adjusted to 10 µm then start cutting till you see the full face of the tissue, then adjust the Microtome to 4 µm and start cutting the ribbon. When getting the best ribbon, they were transfer to the water bath carefully to avoid turning up the ribbon and changing the needed side. The ribbons were not leaved at the water bath for long time to avoid expand the tissue. Once the best section of the ribbon was placed on a slide at the right orientation of the tissue, the slide is labelled with the name of the organ and the date and my name. Then, all the prepared slides were put onto the Heating plate to let them dray for at least 24 hours(1).
5- Staining:
Once the tissues get dried, they were taken to be stained. 6 slides were stained by (H&E), Liver, Kidney, Lung, Oesophagus and trachea, Duodenum and Bone. AS mentioned in (Exercise 1A&1B), H&E stain can be applied by two methods. The first one is Regressive and the second one is Progressive. Mayer’s Haematoxylin is used with the Progressive method, Harris’ Haematoxylin is used with the Regressive method. In this experiment, Regressive method was used to stain the slide because it is rapid and the differentiation can be controlled visually by examination with a microscope (1).
a-One slide of Kidney section was stained by Periodic Acid Schiff (PAS) stain with Diastase Digestion (DD). the staining technique was applied on the section of the kidney to distinguish components of Glycogen from other carbohydrates.
b-One slide of duodenum was stained with Alcian Blue Acid Schiff’s (AB_PAS) to differentiate the Neutral Mucins and Acidic Mucins.
c-One slide of liver was stained with Hall staining method and another liver slide was stained by Perl’s staining method for detecting the presence of bilirubin and hemosiderin in the tissue.
d-One slide that has Oesophagus and trachea was stained by a method that called Masson’s Trichrome staining to evaluate the fibrosis present in tissue by demonstrating collage.
e-8 Control slides was stained by special stains for a compression purpose. The stains that were used are:
1-Hall’s stain to demonstrate the Bilirubin in Liver tissue.
2-Perl’s stain to demonstrate the Hemosiderin in Liver tissue.
3-Congo Red stain to demonstrate the Amyloid in Liver tissue.
4-Periodic Acid Schiff’s(PAS) stain with Diastase Digestion to demonstrate carbohydrates like glycogen in kidney tissue.
5-Alcian Blue Acid Schiff’s (AB-PAS) to demonstrate and differentiate different type different type of Acid mucin in Duodenum.
6-Masson’s Trichrome to demonstrate Collagen in Duodenum.
-Methods:
Fixation and Dissection:
Every Three students shared one Rat:
Procedure refers to histopathology laboratory manual page 29-31(1).
Operation of tissue processing machines , automated strainers and embedding tissue specimens
Procedure refer to RMIT University histopathology Laboratory Manual, Histopathology and Cytopathology 2016, Page 33.
Preparation of paraffin section:
Procedure refers to histopathology laboratory manual page 37- (1).
Embedding:
Procedure refers to histopathology laboratory manual page 36 (1).
H&E staining:
H&E Progressive stain it used for rat sections:
Procedure refers to histopathology laboratory manual page 25-24 (1).
Special staining:
Refers to all special stains procedure in histopathology laboratory manual page 47-62 (1).
a- Identification of amyloid and pigments (Perl’s – Hall’s stains)
procedure refer to RMIT University histopathology Laboratory Manual, Histopathology and Cytopathology 2016, Page 47.
b- Carbohydrates (PAS – PASD- AB/PAS)
procedure refer to RMIT University histopathology Laboratory Manual, Histopathology and Cytopathology 2016, Page 57.
c- Connective tissues (Masson’s trichome stain)
Procedure refer to RMIT University histopathology Laboratory Manual, Histopathology and Cytopathology 2016, Page 67.
Discussion:
During preparing the slides from the beginning of dissecting the rat until putting the cover slip on the slides, some problems were facing and there are many mistakes happened. These mistakes were avoided as much as possible and the one that were not avoided the will be mentioned in the following section.
Kidney:
Detaching the kidney of the rat was not that difficult, because it was not connected with another organ or covered by muscles, so no problem was faced wen detaching the kidney.
Because the wrong sectioning of the kidney there was some difficulties when embedding it. After embedding the section, it was found that the one side of the section was prominent and the other was bogged down in the wax so it will be hard to get the full face when trimming and this issue was solved by re-embedding the section. No problems were faced when cutting the kidney block by the microtome, but because of the position of the microtome which was close to the door, when transferring the ribbon to the water bath, the door was used so some of the good ribbon were flaying away. The kidney slide was stained by H&E regressive method and the Histological characters were clear to recognise. The slide was correctly labelled but there was an excess DPX on the edges of the cover slip.
Lung:
Detaching the lung was a bit difficult because of the rib cage but it was done. No problems happened during embedding the section, it was appropriate in shape and size so it was embedded easily as well as nothing went wrong when using the microtome unless the air that comes from every time it was opened and the all microtomes in the lab were busy so changing the microtome was not an available option. Staining the lung slide was done by the H&E Regressive method and all the Histological features were found comparing to the control slide. An excess DPX was around the cover slip but has no effects on the visibility. The slide was labelled correctly with required information.
Liver:
When detaching the liver of the rat the overflowing of the blood caused a little mass on the bench but the mass was cleaned up otherwise there was nothing difficult. An appropriate three pieces were cut of the liver, washed with saline and put in the fixative reagent. Embedding the liver was easy and the section was put in the centre of the mould. Nothing went wrong when cutting the ribbons of the liver and there were but on the slide at the same orientation of the section as well as the slide had some excess DPX around the cover slip but have no impact when examine the slide microscopically. Hall and Perl’s stains were used to stain this slide. Hall’s stain was used to differentiate Bilirubin and Perl’s stain to differentiate Haemosiderin. Moreover, both stains responded positively, Parl distinguished the Haemosidrin and Hall distinguished the Bilirubin in the tissue. Labelling was done correctly but there was some excess DPX on the edges of the cover slip.
Duodenum:
Finding this organ and dissecting it was a bit difficult because it is small in size and placed beyond the stomach as well as cutting it into three sections was not easy, because it was dissected wrongly. There were some problems happened when embedding the section.
Placing the duodenum section in the middle of the mould was hard because it was sticked to the forceps and the way that the section must be placed is so difficult to be applied but it was nearly done on the right way after several attempts. When trimming and cutting the section, no problems were faced and the ribbons were correctly placed on the slide in the middle and at the same orientation of the section. The slides were correctly labelled and there was an excess DPX around the cover slip. H and E Regressive method was used to stain on of the slide. Comparing to the controls slide the H and E Regressive was an efficient method. The Alcan-PAS stain showed a positive respond and that illustrates the acid mucins and neutral mucins.
Oesophagus and Trachea:
It was so difficult to detach these two organ because of the position of them and they must remain together as required. In addition, cutting these two together into three sections also was not that easy because of the wrong dissecting but it was done correctly. Placing the sections on the right way in the mould during the embedding was extremely challenging because of the size of the section as well as they were two sections must be placed together at the same time. After so many attempts embedding was done at the right way and the block was left on the cool surface to get ready for the cutting step. No problems were faced when cutting the ribbons but the confusing thing was happening when trying to place the selected ribbon on the slide at the same orientation but finally it was done but not at the same orientation. The slide was stained using Regressive H&E method and comparing to the control slide the result was satisfactory. Masson’s Trichrome stain showed a positive response that distinguishes the nuclei stained black, muscle red and the collages blue. Although there was an excess DPX, the Histological characters were clear. The slide was labelled correctly.
Femur:
The femur was the lasted dissected organ because it was so difficult to detach it and remove all the muscle that were covered it. Detaching the femur required a group working because the two rat’s femur was detached to be divided between the three students, so one student was dissecting the femur and the second was remove the surrounding muscle and the third one was taking the femur to the demonstrator to be cut at the appropriate way. The cut femurs were decalcified by the demonstrators and giving back to the students. No problem happened during the embedding process as well as when cutting the sections and put them on the slides. H and E Regressive methods was used to stain the slide and comparing to the controls slide, the stained slides show a satisfactory result. The all histological features appeared clearly and they were recognised although there was an excess DPX around the cover slip. The slides were correctly labelled with the required information.
Conclusion:
The useful of this report is the chance that has been given to take part in every single step of preparing Paraffin section form Rat and reporting the results in appropriate way. In addition, there have been problems that were faced and solved which is giving some experience and confident. All the result seems negative but there are some errors that have no Impact on the visibility under microscope.
3-What are the other clearing agents that could have been used for processing your specimens? Briefly discuss their advantages and disadvantages.
1- Chloroform would be used and because it does not damage the tissue and it is gradual in action, but it might cause health harmful because of the chloroform or the tetrachloride usage (1).
2- The Cedar wood oil could be used. Its advantage is that it is gradually actioning and lacking the significant effect on hardening the tissue section. However, it is expensive and actioning slowly on tissues (1).
3- The Limonene-derivatives have an advantage that it has low toxicity level unlike the xylene, but it has a pungent smell (1).
4-What effects can prolonged decalcification have on tissues?
Choosing an optimal schedule for decalcified bone or other decalcified tissues is so important because if the decalcified tissue over-decalcified with a strong acid decalcifiers that would damage the tissue and spoil the staining of the nuclei and in some cases, it might macerate the tissue elements and makes it soft which make the sectioning process more difficult or even impossible (5).
5-What precautions should be taken when setting and loading a tissue processor?
Because of fresh specimen con be easily damaged, making sure that the specimen has been fixed optimally is so important before starting processing. In addition, making sure that the all reagents are checked out and ready to use. Setting each organ with the suitable programme of processing is one of the most important step in processing, so the tissue that need to be decalcified must be decalcified before starting processing the tissue as well as the others which do not need to be decalcified put in the appropriate place with adjusting the machine to process them as required. The type and the size of the tissue must be considered prior to their placement in the processor.
6-What methods other than a chemical test for calcium ions are available for determining the end point of decalcification? Comment on their reliability.
1- X-ray the specimen is the best way to determine the end point of decalcification, also with a high quality X-ray, the small residual calcium deposited can be revealed as well as it allows further treatment to be requested if required (5).
2- physical tests can be applied to determine the end point of decalcification. Although these tests might be done successfully just with expertise, it is not considered to be reliable and it is requiring trimming of the specimen to feel whether there still calcified areas or not as well as some time manipulation, bending probing are required (5).
7-When embedding tissues, what should be avoided in order to ensure that quality is achieved?
First of all, checking up that the embedding station power on and ready to use as well as the heater and the color surfaces are adjusted at the optimal temperature. In addition, making sure that the embedding machine is full of paraffin wax and it is kept at the appropriate temperature (4).
1- putting the specimen in the wrong mould size must be avoided, so the size of the mould should be chosen regarding the size of the specimen
2- avoiding using the uncleaned forceps must be considered to avoid sticking the specimen to the forceps as well as it will be placed at the wrong side, and avoid leaving the forceps out of their place to not get cold and they will not be ready to use.
3- facing the cut side up must be avoided because the cut side must be always faced down
4- When placing the specimen into the mould avoiding leaving the mould on the cool spot for long time before putting the cassettes on the specimen and fill it again with wax to avoid forming a buffer zone between the wax that was put to settle the specimen and the one which has been filled later as the block will be as two blocks sticking together and that will impact the sectioning process badly and might even damage the block.
5- taking the paraffin blocks away from the cooling area must be avoided, so the blocks must remain on the cooling area enough time to get solidified.
References:
1- Danks J. Week 5: Staining principles [Lecture notes]. MEDS2106/2111: Histopathology 1. Melbourne: RMIT University; 2016.
2. Histopathology Laboratory Manual 2015. Melbourne: RMIT University; 2016.
3. Rolls G, 2016, An Introduction to Specimen Processing, viewed 27 Sep 2016, http://www.leicabiosystems.com/pathologyleaders/an-introduction-to-specimen-processing/
4. Rolls G, 2016, Steps to Better Processing and Embedding, viewed 27 Sep 2016, http://www.leicabiosystems.com/pathologyleaders/steps-to-better-processing-and-embedding/
5. Rolls G, 2016, An Introduction to Decalcification, viewed 27 Sep 2016, http://www.leicabiosystems.com/pathologyleaders/an-introduction-to-decalcification/
6. Lab.anhb.uwa.edu.au. Blue Histology, Viewed cited 27 October 2016, Available from: http://www.lab.anhb.uwa.edu.au/mb140/.
