Lab 2 Immunocytochemistry
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BMB 210 Lab 2
LABORATORY 2
IMMUNOCYTOCHEMISTRY - PART 2
Before lecture, start the rinsing procedure as described on page 2, # 1&2.
In today’s lab, you will complete the immunocytochemistry experiment that you began last week.
INTRODUCTION
Recall that in last week’s lab, you began the incubation of rat pancreas sections with rabbit anti-insulin and mouse anti-glucagon primary antibodies. Today, after rinsing the sections
in KPBS to remove unbound primary antibodies, you will add the secondary antibody solution. Two different secondary antibodies will be used: donkey anti-rabbit and donkey anti-mouse. These antibodies were generated by injecting donkeys with either rabbit or mouse antibody proteins. The donkey immune system produced antibodies against the foreign proteins, and these donkey antibodies will be used as the secondary antibodies that bind to the primary antibodies
. Several secondary antibodies can bind to each primary antibody (see Fig. 1). Antibodies are not visible through the microscope unless they are tagged with some type of marker. Covalently attached to the secondary antibodies are fluorescent molecules that can be seen using a fluorescence microscope. These fluorescent molecules contain electrons that are excited by certain wavelengths of light. As the excited electrons return to their ground state, photons of a specific wavelength are emitted and are seen as a particular color. The fluorescence microscope contains filters that allow the passage of certain wavelengths and not others. The donkey anti-rabbit secondary antibodies are tagged with A488, which fluoresces green. The donkey anti-mouse secondary antibodies are linked to Rhodamine Red-X, which fluoresces red. Cells producing insulin or glucagon, therefore, can be seen through the microscope via a series of antibodies and fluorescent markers. What color should insulin-
producing vs. glucagon-producing cells appear?
Secondary antibodies (donkey anti-rabbit or anti-mouse)
with fluorescent markers attached
Primary antibody (rabbit anti-insulin or mouse anti-
glucagon)
Antigen molecule in tissue (insulin or glucagon)
Fig.1. Diagram illustrating the consecutive binding of primary and secondary antibodies to an antigen in a tissue.
Page 1 of 3
BMB 210 Lab 2
EXPERIMENTAL PROCEDURE – Wear Gloves!
Secondary Antibody Incubation
1. Remove the slides from the humid chamber one at a time. Turn the slide on end to allow the
puddle of primary antibody to run onto a paper towel, and place the slide in a slide carrier inside a staining boat filled with 1X KPBS. Do not let the slides dry.
2. Rinse the slides in 1X KPBS two times for 5 minutes each rinse (2 X 5). After the final slide is put into KPBS from step 1, begin timing. This is rinse #1. After 5 minutes, gently remove the slide carrier and pour the KPBS into the sink. Pour fresh 1X KPBS into the boat and return the slide carrier to the boat. This is rinse #2.
3. After the second 5 minute rinse, remove the slides one at a time, wipe the excess KPBS from the back with a Kimwipe as you did last week, and set the slide in the humid chamber. Add 100
l of secondary antibody solution onto the tissue inside the hydrophobic/PAP ring. Repeat for all slides.
DO NOT LET THE TISSUES DRY! The secondary antibody solution contains donkey anti-rabbit linked to a green marker, A488 (A488 D
R) and donkey anti-mouse linked to Rhodamine Red-X (RedX D
Ms). The secondary antibody was diluted in the same dilution buffer used last week.
4. Cover the humid chamber and let the slides incubate at room temperature for at least 1 hour.
***During the 1-hour incubation, we will complete the primary research article citation and reference formatting activity.***
Cover slipping and observation under the microscope
After the slides have incubated for one hour:
5. Remove one slide at a time from the humid chamber, and blot the secondary antibody solution onto a paper towel as you did above. Rinse the slides in 1X KPBS, 2 X 5, as described in steps #1 and 2 above.
6. Coverslip with an aqueous mounting medium: After the last rinse, remove one slide at a time and lay it on a paper towel -- specimen side up! Using a Pasteur pipette, add 2 drops of mounting medium (1:1 KPBS/glycerol) over the tissue on the slide. Coverslip with a 24 X 40 mm
coverslip, avoiding air bubbles as much as possible.
7. Lay the slides horizontally in a plastic slide tray. When handling the slides, always hold them horizontally or the coverslip will slide off. Your instructor will take you to the fluorescence microscope in the “imaging room” in Y103 to look at your slides.
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Please help with questions 2, 3 and 4 of part four
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VILION
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Quiz Submissions - Take: Unit D Practice Quiz
JUDITH BIRECH (username: nj2191ne)
Attempt 1
Written: Feb 9, 2023 2:20 PM - Feb 9, 2023 3:06 PM
Submission View
Released: Feb 13, 2023 12:01 AM
Question 2
A patient had decreased mast cells. Which do you predict?
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a) decreased histamine & increased inflammation
Ob) increased histamine & increased inflammation
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Procedure:
1. Prepare 5 test tubes. Place 1 ml of 1% starch and add 10 drops of saliva to
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tube leave at room temperature, the 3rd
tube in 40°C , the 4th
tube at 60°Cwater bath and the 5th
tube boil for 2
minutes..
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tube
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4. Test the contents of each tube with iodine and benedict’s tests.
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https://www.studocu.com/en-us/
document/university-of-arkansas/microbiology/other/immunology-lab-worksheet-student/7960193/view
Questions from ELISA Simulation Introduction Document (posted in Lab Module 8 folder):
1. What term is the word “antigen” derived from?
2. Where are antibodies found in the body?
Plasma of the blood
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enzyme-linked immunosorbent assay
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Calculate the dilution factors for each tube.
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Calculate the total number of cells in tube number 2.
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concentrations (ug/ml) of the antibiotic Oxacillin in them. From
Left to Right, the concentration of Oxacillin is going
down
acillin concentration, pg/ml
64 32 16 8
4
2
1 0.5 0.25 0.13 0.06 0
SA
dn
The same amount of S. aureus broth culture is added to each
well and the tray is incubated. A color change shows that S.
aureus grew in a given well.
MRSA
What is the lowest concentration at which SA is inhibited (does
not grow)?
64mg/ml
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What is the MIC for Oxacillin vs MRSA?
- Notes
Comments
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It was important for Lilly to understand how the bacteria were able to cause disease in patients.
The mechanism of pathogenesis by M. tuberculosis starts in the lung alveoli. The cell wall of M.
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infect other macrophages. As the cycle of infection slowly progresses, the body's response to the
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surrounding the bacteria to restrict its spread). Eventually, the bacteria can escape the granuloma
and infect other parts of the lung.
Transmission of Mtb
Initial infection and
Granuloma cavitationy
replication of Mtb
in macrophages
and dissemination
of Mtb in the lung
Infected
macrophages
Caseating
granuloma
Recruitment of
Innate and
adaptive immune
cells
Infected cells undergo
necrosis resulting in
the…
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OO HUAWEI Nova 2 Plus
DUAL CAMERA
Question
39
Regardless on the method of generating the vaccine or its type, there is a common fact which is?
Select one:
Not yet
O a.
The immune system will produce specific and determinant antibody for each virus
answered
Marked out of
O b. The immune system will not react to viruses but it will react to bacterial infections only
1.00
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O C.
The immune system will produce several antibodies for one type of virus
question
O d. The immune system will produce a general antibody to any invading virus
e.
The immune system will engulf any virus
Question
40
HETEROCHROMATIN refers to
O a.
Regions of the genome that is unfavorable for gene translation
Not yet
answered
Ob. Region of the genome that is favorable for gene replication
Marked out of
O C.
Regions of the genome that is favorable for gene transcription
1.00
Od. Regions of the genome that is unfavorable for gene transcription
P Flag
question
O e.
Regions of the genome that is favorable for gene…
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