9.9.1 Estrogen receptor α (ERα)
The antibody used was the monoclonal estrogen receptor alpha antibody (MAB-443) from Affinity Millipore. The protocol is shown in Tab. 30.
Table 30: Protocol for the immunohistochemical staining of the Estrogen receptor α (ERα)
Duration Reagents/Treatment
3 x 10 min Xylene
2 x 5 min 100 % alcohol
2 x 5 min 96 % alcohol
2 x 5 min 70 % alcohol
2 x 5 min Distilled wate
Preheating of TEC buffer at 96-99°C
30 min Antigen unmasking in TEC buffer, water bath 20 min Cool down in TEC buffer
10 min 2 % H2O2 in Distilled water
5 min Rinse in TBS
20 min Incubation with normal horse serum 10 min Incubation with free protein serum
Over night Incubation with first antibody 1:200 in FPS
5 min Rinse in TBS
20 min Incubation with second antibody
5 min Rinse in TBS
3,5 min Incubation with DAB
1 x 5 min Rinse in TBS
15 min Rinse with tap water
20 seconds 70% Alcohol
20 seconds 96% Alcohol
2 min 100% alcohol
2 x 5 min Xylene
Mounting with Eukitt
9.9.2 Progesterone receptor (PR)
The antibody used was the monoclonal progesterone receptor antibody (MA 1-410) from Affinity BioReagents Inc., Golden, CA. The protocol is shown in Tab. 31.
Table 31: Protocol for the immunohistochemical staining of the progesterone receptor (PR).
Preheating of EDTA buffer at 96-99°C
20 min Antigen unmasking in EDTA- Puffer, water bath
20 min Cool down of EDTA buffer
3 x 5 min Rinse in PBS
20 min Incubation with normal goat serum Over night
Incubation with first antibody 1:200 + 1.5 % BSA dilution in PBS
3 x 5 Min Rinse in PBS
30 min Incubation with second antibody
3 x 5 Min Rinse in PBS
5 min Incubation with DAB
1 x 5 min Rinse in PBS
15 seconds Nuclear staining with hematoxylin
15 min Blueing with tap water
2 min 70% alcohol
9.9.3 Oxytocin receptor (OTR)
The antibody used was the polyclonal oxytocin receptor antibody (LS- A244) from LifeSpan Biosciences, Inc. The protocol is shown in Tab. 32.
Table 32: Protocol for the immunohistochemical staining of the oxytocin receptor (OTR).
20 min Antigen unmasking in citrat buffer, water bath 20 min Cool down in citrat buffer
3 x 5 Min Rinse in TBS
15 min Tween (0,1 %) in TBS
3 x 5 min Rinse in TBS
20 min Incubation with normal goat serum Over night
incubation with first antibody 1:200 + 1.5 % BSA dilution in TBS
3 x 5 min Rinse in TBS
30 min Incubation with second antibody 3 x 5 min Rinse in TBS
9.9.4 Prostaglandin F2α receptor (FPR)
The antibody used was the Polyclonal PGF2α receptor antibody from Cayman chemical company, Cat # 101802 (Tab. 33).
Table 33: Protocol for the immunohistochemical staining of the prostaglandin F2α
receptor (FPR).
Duration Reagents/Treatment
2 x 10 min Xylene
2 min Absolute alcohol
30 min 80 % alcohol + 30% H2O2 (196 mL + 4 mL)
2 min 70 % alcohol
3 x 5 min Rinse in PBS pH 7.2
Preheating of citrate buffer at 96-99°C 20 min Antigen retrieval in citrate buffer, water bath 20 min Cool down in citrate puffer
3 x 5 min Rinse in PBS
20 min Incubation with normal goat serum
Over night Incubation with first antibody 1:1000 + 1.5 % BSA dilution in PBS
3 x 5 min Rinse in PBS
30 min Incubation with second antibody
3 x 5 min Rinse in PBS
5 min Incubation with DAB
1 x 5 min Rinse in PBS
10 min Rinse with tap water
2 min 70% Alcohol
2 min 80 % Alcohol
2 min Absolute Alcohol
2 min Isopropanol
2 x 5 min Xylene
Mounting with Eukitt
Acknowledgments
The completion of this work is the result of the effort and support of many people.
Although I can only mention a few of them in these lines, know that words can barely express the gratitude and respect that I feel for all of them.
Firstly, I would like to express my most grateful acknowledgments to all the people who provided the scientific and financial support that made this work possible. I must thank Prof. Bollwein for the opportunity to perform this doctoral thesis and for all the advice, patience, and knowledge afforded throughout the program and Prof. Pfarrer for generously providing access to the facilities of the Anatomical Institute. Her invaluable suggestions, support and knowledge of the bovine uterus has enriched the quality of this work, and for all that I am deeply grateful.
I am particularly indebted to Dr. Heppelmann. She has gone beyond the call of duty to help me achieve my goals through the most careful supervision, understanding and encouragement. Without her brilliant ideas, her knowledge of bovine medicine and biostatistics and especially without her comforting words in some hopeless moments, this work would have not been possible. Furthermore, I have to thank her for opening the door for me to the fascinating world of science.
I would like to express greatest thanks to Marion Gähle, Doris Walter, Christel Hettel, Katharina Baur and Sandra Wilkening for their invaluable advice and assistance, and to Marcelo Gil Araujo for his help with statistics. A special thanks to Gregory Bodkin and Hendrike Knaack for their advice in English and German, respectively. For their help during my duties and experiments, I have to thank the animal keepers and the assistants of the Clinic. My sincere thanks go to Karoline Krach and Korina Kedves for their support during the experimental of the project. I would like to especially acknowledge Lars Holzhausen for his patience during all the long nights in front of
the monitor, and for always having ready a smile and a comment to turn weariness into motivation. Very special thanks go to Julia Volland for every step that we shared in this long-distance run which has paved the way for a friendship I can only be proud of; and to Nilay Yucesoy for her never-ending goodness and everlasting disposition to help and care.
My warmest gratitude is for my Hanoverian family, Hakan Gürler, Letizia Debertolis, Burcu Cinar, Petra Kruse, Guillermo Vizuete Calero, Ioannis Proios, George Niozas, Cristina Andreu Vázquez, Claudia Raschka, Marie Meyerholz, Kirsten Mense, Mimi Hörnig and all the doctoral candidates from the Clinic of Cattle who transformed “Doc Zimmer” into a sweet second home. They shared my good and my bad days and we made from these years a memorable experience. For sweetening those long summer evenings in front of the computer with nice discussions, stories and candies, I have to especially thank José Antonio Infantes Lorenzo, Sofía Stirling, Álvaro Gutiérrez Bautista, Jesús Maldonado and Ana Belén Ruiz Martínez. I am totally convinced of the wonderful careers that await all of you, and I am just happy to be a part of it.
Last but by no means least, I would like to thank my parents for all their support and love. They cannot imagine the extent of my gratitude for always encouraging me to follow my dreams and for believing in me. Through their long-distance care, I always felt at home even though I was so far away. Very special acknowledgments go to my hard-working sister, Raquel, for always being willing to listen to my worries and problems, and for always finding solutions to them. I would also thank my close relatives, especially my cousins and my aunt Isabel for continuously asking about my work and my life in far and cold Germany. I would like to express my gratitude to Dimitri Radko, for being a great companion in both work and life. I had the honor to share these years of scientific and veterinarian work with an old friend. I thank Letizia Debertolis for so many moments that transformed this phase in a precious life. To my